Nyepi Day (Silent Day) In Bali

Silent Day, the religious holiday, is the only one which is happening in the world. There is no other similar day anywhere else but Bali. Traditional Hindhu’s celebration falls every year according to Hindhu ancient calendar. This special day is celebrated every March in a year. It is a symbolic act for the winning of good (Dharma) over bad energy (Adharma) and also marking Balinese New Year based on their traditional calendar. During the Pengrupukan, all Balinese do such a small ceremony to dismiss evil spirit around their compound and also the area they live usually at the junctions. According to Hindhu’s sacred book, during this day, all bad energy or evil spirits are freely wandering in the world. Exorcist ceremonies are driven away those spirits for not to disturb the humans live.

All family members carry dried coconut leaves which are burnt and make noisy sounds out of kitchen tools. Then they will go around the house while carrying the burning leaves and the noise to each room, toilet, kitchen area, and every corners of the compound to purify the living areas including the junctions. This activity takes place during the sunset or Sandi Kala, the time for the bad spirits raise up. When the night comes, every village in Bali does the next procession which is carrying deity representations around the village. The deities are usually in form of furious and horrible appearance.

However, other symbols in form of PUNK model, Motor bike driver and other modern forms are adopted. The gigantic monsters representation is carried by the young people while the followers which are the rest of the villagers carry torches made from bamboo. After carrying this monstrous symbol, then it will be burnt down at the junctions or even in the grave yard, to end the Pengrupukan Night. The next day which is the due date of Nyepi Day, all activities are totally stopped as if the world has been stopped from moving. There is nothing on the road, no cars or vehicles, no people wandering around, no lights at night, no TV or radio, industries and offices are stop operated including the airport. The day of silent starts from 6am to the next morning, 6 am. The whole Hindhu are fasting and meditating during the Nyepi day for self purification. Additionally, there are four things should not be done; which are: no lights, no work, no entertainment and no traveling. Other religions are also participating by doing those four. They show their respect to live side by side in the peace. Tourisms aspects are also showing the same respect by not using lights and allowing their guests coming down to the road. The next day, Ngembak Geni, is highlighting the end of the moment of Silence. The live gets back to normal again. All aspects of live runs back to normal routine till the next Nyepi day come again.

It is one of the region’s most popular beaches and is dotted with myriads of seafood restaurants and scores of beachside cafes. The sandy beach lacks dangerous rocks and has become a haunt among surfing enthusiasts. Sunbathing, shopping and partying are the must-do things at Kuta. Nestled in the middle of Kuta and the popular hangout, Seminyak, Legian Beach is an apt place to indulge in flurry of activities including shopping, swimming, meditating, exercising and spa. The beach makes for an ideal spot for sybarites as many posh hotels accentuate the grace of this beach. Suffused with perfect party ambience and replete with countless bars and lounges, the beach has become a popular party destination among many tourists netting air tickets to Bali.

Bask in night revelry and gorge delicious dishes offered by beachside restaurants! Another popular beach that shows on itineraries of most travellers grabbing tickets to Bali is the Uluwatu. It is located on the western shores of Bukit Peninsula on the southern coast of Bali. Revered as a perfect surfing spot for experienced surfers and discerning epicureans, Uluwatu offers number of restaurants to sample the pleasing seafood and enjoy a chilling sundowner. One can also take delight in kite boarding and snorkelling. The ancient Pura Luhur Uluwatu temple, located near the beach, is one of the oldest temples in Bali and a centre of attraction that beguiles visitor with its legendary setting.

The island of Bali is located in the tropics, just about 6º South of the equator. Blessed with warm temperatures, where the variation is very small throughout the year, with sunrises at 6 am in the morning and sunsets at 6 pm. Gentle climate, friendly people and modern Villas in Bali, make this place a paradise on earth. When planning your next vacation consider the Saner beach, situated in the southeastern side of Bali, and just a 10-minute drive from Denpasar. Bali Villas located in this area are excellent to jog on the sandy beach and watch the sunrises. Many of those villas near the ancient temple were constructed as the first resort centers developed on the island.

Nusa Dua is the newest luxurious resort area on the island, where you are more likely to find the most sumptuous Villas in Bali, including hotels, condos and other world-class facilities. The crystal clear water of this beach provides excellent diving and snorkeling, featuring also waves on the northern and southern area of the beach for surfing. People wanting to rent a Villa in Bali closer to a romantic setting, the Soka Beach in the northwest of Denpasar, offers a small quiet beach, located on the way to Gilimanuk, the ferry port town connecting Bali and Java. Nearby, and along the path towards Gilimanuk, the Medewi Beach in the village of Pulukan enriches the spirit and soul of locals and visitors. Lovina Beach is near the northern tip of the island that lays a stretch of Bali Villas by the Bali Sea, with excellent snorkeling and diving, as well as chartered boats to go out into the sea. Sunset watching approaches paradise from here, a beach not as crowded as the Kuta Beach. Last but not least on your list of Villas in Bali, are those located in Jimbaran, which offer the convenience of being located south of the airport.

Because most of the beaches on the island of Bali have such incredible surf and waves, this does not make them ideal for snorkelling. However, if you travel to the east of the island to Padangbai, you will find three beaches with paradise white sand and calm, clear waters. This quiet sleepy fishing village doesn’t attract so many visitors, meaning the beaches are often quiet and peaceful. Bloo Lagoon beach, located near the Bloo Lagoon eco-resort just over the hill to the left, has the best snorkelling, with calm clear waters, scattered rocks and coral. Amongst the sea life you can expect to see Napolean Wrasse, reef sharks, stonefish, moray, rays, squid, octopus and blue ribbon eels.

There is also a beautiful, secluded white sand beach over the hill to the right of the town, which is lined with palm trees and is the closest thing to a tropical island paradise. If you’re looking for a beach full of beautiful people, with plenty of bars and restaurants and activities going on, then Seminyak is the beach for you. Whilst the long sandy beach isn’t as idyllic as some of the others on the island, it is great for people watching, attracting hundreds of tourists looking for fun in the sun. Seminyak’s beach is an extension of Kuta Beach and is a long stretch of flat, compact sand with rolling waves perfect for learning to surf or having a go on a boogie board.

You can lie back on a sun lounger with a Bintang or if you’re feeling a bit more energetic, play a game of bat and ball. Seminyak’s beach has one of the best views of the sunset, so if you can afford the price tag, have a cocktail and watch the sun go down from Ku De Ta, Bali’s world-famous bar. Few backpackers have heard of this beach, but Balangan, down in the south of the island past Jimbaran, is one of the ultimate for relaxing and letting your cares fade away. This sandy beach is lined with palm trees and is also a great spot for skilled surfers, with glassy waves rolling in over the coral reef.

You can relax on a sun lounger in front of one of the beach shacks with a banana milkshake and a view of the surf, or go for a swim in one of the many natural rock pools. As the tide goes out you can go for a wander over the reef to get a closer view of the surfers in action. Balangan is a peaceful, tropical beach which will make you feel like you’re a million miles away from reality. Uluwatu, at the southern tip of Bali, is famous for its temple perched dramatically on the cliffs. Tourists flock to Uluwatu to see the band of monkeys that run around the temple complex, and to witness the traditional dance performance at sunset. But Uluwatu is also the most famous spot in Bali for experienced surfers, offering perfect waves during high season when the swell is good. Pantai Suluban is sheltered by steep cliffs, so on a windy day it’s perfect if you want to avoid getting sand in your eyes. Set in the cliffs are several warungs serving traditional Indonesian food like Nasi Goreng and banana pancakes, as well as a couple of ding repair shops and shops selling surf wear. Sit and enjoy the incredible views with a spot of lunch or climb down the steep steps to the shingle beach where you’ll find cool caves and rock pools.

There is little that you won’t find to do in Bali when you think of a tropical island paradise. The beach is definitely very popular, but it is when you head into the water that you begin to actually experience all that Bali has to give. Tours round the island will include more popular stuff like snorkeling, scuba diving and fishing trips, but there are also some more unusual underwater activities available. Another highly popular water activity in Bali is whitewater rafting that offers tours for kids of five years of age and all of the s are very well set up and adhere to world safety standards. Apart from the water sports, there are other inland tours that are just as exciting and will get your heart racing.

Touring the volcanoes that stretch from the east coast to the west coast can be stirring, particularly when you have reached the summit and jump off with your para glider. Many paragliding tours are in operation as a result of the abundant areas that are available to launch from. If flying through the air isn’t for you, visit the elephant safari park renowned as the best one in the world. They offer short rides for kids as well as long treks through the Balinese countryside sitting on the back of your own big elephant. It might not appear as much fun as some of the other activities, but once you climb up on top of a seven foot elephant, your heart will skip a beat or two. If all these activities seem too much then you can take your own tour at your own pace and do a Bali shopping tour that will be as exciting as anything else. One of the great things about Bali is that it never disappoints, there’s always something that will leave you awestruck and left with memories for a whole life.

Many Balinese live by the concept of Tri Hita Karana—broadly, harmony with people, nature and God—and David Bowie requested that his body be cremated in a Balinese ngaben ceremony. This ever-present spirituality is celebrated at Ubud’s Bali Spirit Festival, a mass love-in at the wood-and-thatch Bali Purnati Arts Centre. Everyone knows Ubud gives good spirituality (like, duh). But it’s also got one of Bali’s most forward-thinking food scenes, typified by Noma-ish Locavore, where the only thing that’s imported is smiling Dutch chef Eelke Plasmeijer. Mozaic, will convene at local favorite Mozaic to create a special five-course menu. The big hitters of the World Surf League make only one stop a year in Asia, when they rock up to compete in the Corona Bali Pro Event entertainment at Keramas’s Komune resort, on the southeast coast. Last year, Brazil’s Italo Ferreira won the competition on the famous barreling wave, watched by crowds drinking cocktails and taking dips in the Komune Beach Club’s infinity pool. Sports viewing doesn’t get much more civilized.

The reason why tourist from many countries coming to Bali probably is the sea, sand and sun of the Bali Beaches. There are so many beautiful beaches like Kuta, Sanur, Nusa Dua, Uluwatu, Jimbaran, Dreamland, Candi Dasa, Lovina, Tanah Lot, Nusa Lembongan and Nusa Penida with their beauty panoramic view. Kuta is the most popular beaches, accessible, lively and commercialized of Bali ‘s southern beaches. On the south, the beach is fenced by the airport’s runway, which gives you a breathtaking landing experience. The beach stretches as far north as the eyes can see. Kuta is well known for its long sweep of white sand and is a big draw for those who want to test their surfing skills, boogie surf, playing football, with friends or just sea side walk and seeing people.

There are so many events held here such as Kuta Carnivals, Surfing Competition and Volley Ball competition. But Kuta is more for surfing than for swimming and more for parties than for sunbathing, and a visit to Bali is not complete without visiting Kuta Beach and seeing its stunning sunset. There are so many expatriates and residents coming to this beach. Normally they swim, sun-bathe, play football or just relax and order some drinks at the restaurants around this white sand beach. At this area, there are some restaurants, bars and clubs, where people can dance till the morning come. There is a temple at this beach and called ‘Pura Petitenget’ that’s why this beach is called Petitenget.

With grey sand, the Petitenget Beach looks very mysterious, tourists rarely come to this beach only the local people come for swimming or playing. Tuban Beach is just five minutes from the airport and from the famous Legian Street. It has white sand and surrounded by five Star hotels between Kuta and the airport. It’s safe to swim here, as the hotels’ lifeguards will put up flags if there are any rips. This is a good place to grab a deck chair and a good book and enjoying the beautiful sunset. This exotic beach is set in a sort of cave with cliffs looming above. Uluwatu is famous for its waves that can peel perfectly for hundreds of meters over reef, and are reserved for expert surfers only.

Uluwatu has a beautiful sunset that you can see from the top of the hill and you can see the turtles swimming also. However, swimming is inadvisable here. Just down the road from Uluwatu is Padang-Padang, which is another enchanting beach nestled into a pocket of lava-rocks, with an entrance to the beach though a cave crevice. This small cove offers famous world-class waves that attract surfers from all over the world. The water is also safe for swimming and snorkeling. The beach is small but the setting is enchanting and memorable, and like Uluwatu has warungs for drinks and snacks.

Down the Peninsula from Padang-Padang, this long stretch of white-sand beach is free of warungs and people, and still maintaining its original natural make-up of flora along the towering cliffs. Unfortunately, at high tide the beach all but disappears under the waves, and for this reason Impossibles is not an ideal place for relaxing on the sand, although it is a great setting for a sea-side walk at low tide. Swimming is not advised here, and the waves are for experienced surfers only, as they break over reef. Within paddling reach of Impossibles is Pantai Bingin, but this beach totes an extremely different atmosphere.

It is still very natural and quiet, with few people, although there are quite a number of bungalows and a few warungs in the vicinity. This white-sand beach is pockmarked with rock and reef, forming pools in low tide, while at high tide the beach is almost consumed by the sea. Experienced surfers love to come here as the waves are near perfect and often form pristine tubes. The water is also fairly safe for swimming and snorkeling as the waves break on a reef about fifty to one hundred meters from the shoreline. A few hundred meters up from Bingin is Dreamland, which overlooks every other Bukit beach all the way to Uluwatu. Jimbaran Beach is a sleepy cove where fleets of fishing boats colour the scene.

With a slower pace than Kuta and a relaxed friendly atmosphere, the shores of Jimbaran provide a peaceful respite. The perfectly shaped bay is also spared the crashing waves of nearby beaches, providing a safe haven for leisurely swimming. Jimbaran is a popular spot for windsurfing and sailing small craft. Jimbaran’s grey sand and calm waters are attracting more people but the beach still has a sleepy feeling. It is also popular spot for watching the sunset and eating the fresh seafood. It’s about 3 kilometers, around 10 minutes from the Ngurah Rai airport. Kedonganan is a fishing village, where you can see a traditional fish market and Jukung, the traditional fishing boat which present an interesting view. Here, you can order the seafood grilled at the seafood restaurant around the beach while you watch the sun go down the valley.

Nusa Dua, a beautiful stretch of white sandy beach that offers excellent swimming conditions in a protected lagoon, was designed for the luxury conscious with big name hotels lining the beautiful white sand beach. Some of the resorts have private beaches but most areas are accessible. The stretch past the Hilton Resort has been a public beach and during the wet season, the outer reef there is a popular surf break. On the way to Nikko Hotel, after Bali Golf, there is a valley which end in Geger Beach. At the Geger Beach, you can see the many beautiful fishes swimming freely between coral reef and the fisherman in the sea grass field. Geger Beach is quiet s beach where you can swim in the clear water, do fishing, and lay down for sunbathing until the sun go down.

Sanur Beach is the best place to watch the sunrise in the morning. Stretch along way from behind Grand Bali Beach hotel, Sindhu Beach, Pantai Jerman to Semawang Beach. A beach walking making it possible to enjoy the many hotels and restaurants along the strip and enjoy the local snack like spring roll, fried tofu, grill corn and many other snacks. Sanur is a calm, quiet lagoon that offers great conditions for wind surfing and para-sailing. Sanur has a lively history and is still a revered area. The beach often fills up with brightly clad ceremonial folk honoring various auspicious days.

The reef off Sanur is a popular surf break but only works under limited conditions. Many water sport companies are based here, offering a number of recreational activities. It is just 5 minutes from the beautiful Sanur Beach. It is black sandy beach with most of local people come here in the early morning to watch the sunrise and see the Mount Agung. The white sandy beach, is a popular place for surfing, includes the warung and accommodations. In the morning, you can see the beauty of the sunrise, also the Mount Agung can be seen from this beach. And in the middle of the year, The Bali Kite Festival is always held at this beach.

There are various water sports or sailing on the Jukung the traditional boats can be done at this beach. On the black sand, you can have picnic with your friends or family and enjoy the grilled sea food. Padang Bai, a picturesque bay surrounded by majestic cliffs and hills, is a perfect escape at the east of Bali. It is famous for its pure white sandy beach and lovely harbor scenery as a transit harbor to Nusa Penida and Lombok Island. Candi Dasa is the gateway to east Bali. The beach has been mostly washed away due to the heavy coral picking.

This has now been stopped and tidal walls have been erected in an effort to restore the beach. Slowly the coral is returning, Candi Dasa now has become a popular tourist destination. Unfortunately the beach is not suitable for swimming. Amed, around 30 minutes drive from Candidasa, is one of the best scuba diving and snorkeling spots in Bali with its breathtaking under water world. Amed is a traditional cultural center with only minor influences from tourism, you can see the village boys and girls practicing traditional music and the fisherman sail their outrigger canoes out to catch the fish.

Endnote Cheet Sheet

EndNote Cheet Sheet

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Arabidopsis Seed Collection

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Arabidopsis Seed Collection

Luca Comai has devised a simple seed collector suitable for high density use (many plants in a small space). It is effective, very cheap, and easy to construct. The instructions for constructing it are given below. They may sound complicated but the collector is really very simple. Feel free to ask questions or send comments.

 

Seed collector II

Introduction

This seed collector is suited for harvesting seed from individual plants grown in small pots (e.g.: square, 6 cm wide at top, tapered to 4.5 cm at bottom, 8 cm high). Each pot can be placed next to other pots to achieve high density spacing.

 

Materials

  1. Adhesive tape, such as VWR or Time-Med pressure sensitive labeling tape 1.8 cm wide. The tape type is not important as long as it sticks and it holds up to greenhouse conditions
  2. Paper stapler
  3. Plastic film, such as Mylar overhead transparency film (0.002 mil, Vu-Color ). The choice of this film is based on characteristics and availability. We recycle the film used for lecturing. Other types maybe suitable: experimenting is the best way to find out whether the film has the right flexibility. Mylar has one disadvantage: it builds an electrostatic charge that attracts seeds. However, in our case, the film comes for free and it looks pretty with all the lecture notes. We prefer instructors who use multicolored pens.

Construction

  1. Cut sheets 12 cm x 42 cm. Roll them lengthwise on a dowel 33 mm in diameter and 50 cm long. The long sides will overlap by about 1.3 cm
  2. Tape the resulting plastic tube once, at one third the distance from one extremity. Make a continuous ring of tape for maximum strength
  3. Flatten and fold back the end of the tube most distant from the tape, in such a way that the seam is central and internal to the fold. Staple the sides just above the fold. The fold line should be 2 cm from the end
  4. About 9 cm above the fold, make a cut perpendicular to the tube. The cut will comprise half or slightly less than half of the circumference and will place the seam in the center of the cut. The collector is finished
  5. Appress the flattened end of the collector to the side of the pot. Place the cut about 5 cm above the rim of the pot and facing the pot. Tape the collector to the pot with a full ring of tape
  6. Gently spread the cut and introduce the young inflorescence. As secondary inflorescences are produced guide them in the collector or remove them
  7. A plant with a fully developed inflorescence can be easily fitted with a collector.
    • Gently lay the pot on its side so that the inflorescence fits over half a sheet of standard printer paper (7.5 cm x 26 cm)
    • Roll the paper to enclose the inflorescence. Make the roll’s diameter smaller than the collector’s. Tape the roll to avoid unfolding and pull it to where the length of the inflorescence-paper roll assembly is longer than that of the collector
    • Place the pot at the edge of a table with the inflorescence leaning out. Gently push the rolled inflorescence into the collector. Once fully inserted, the tip of the paper roll should stick out or be easily reached. Pull the paper roll out, leaving the inflorescence in the collector
    • Tape the collector as in “5”

Comments

The dimension of the collector can be changed to fit any square pot. Its design can also be modified to fit special situations. We harvest the seed by cutting the inflorescence at its base, throwing the label into the collector and closing its ends. Collectors can be washed and reassembled or thrown away. I would appreciate knowing of any improvement. For big pots and when space is not a limitation, our previous seed collector (see Compleat guide, AATDB) works well.

Dna Extraction Procedure From Human Blood

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1. Draw 5 ml of blood using lavender-top Vacutainer (Beckton-Dickinson; EDTA anticoagulant)
2. Keep cool until prep is performed (e.g. in an ice chest), but DO NOT FREEZE. Highest yield will be achieved by extracting within 24 hours.
3. Empty blood into a 15 ml Falcon tube; add 10 ml of Red Cell Lysis Buffer (RCLB) and mix completely by inversion.
RCLB: 1 mM NH4HCO3
115 mM NH4Cl
Note: You can bring pre-weighed NH4Cl and a 1M solution of NH4HCO3, and use the cleanest water available to make RCLB on-site. Try to use distilled water, but in a pinch I suspect that water from a personal filter (e.g. Pur, etc.) would work. If it is to be used immediately, this solution does not need to be autoclaved; if you plan to store it for more than a day, autoclave.
4. Spin 10 minutes @ 1,200g in a clinical centrifuge.
5. Discard supernatant and resuspend cell pellet in 10 ml RCLB; repeat step 4.
6. Discard supernatant; resuspend cell pellet (it should be white now) in 1.8 ml of White Cell Lysis Buffer (WCLB). It should be extremely gelatinous – like snot.
WCLB: 100 mM Tris-Cl (pH 7.6)
40 mM EDTA (pH 8.0)
50 mM NaCl
0.2% SDS
0.05% Sodium azide
Note: This solution (before the addition of SDS) definitely needs to be autoclaved – this means bringing a heavy, well-protected bottle with you, or being completely certain that you will be able to autoclave on site. Add SDS after the autoclaved solution cools. Remember that the DNA is going to sit around in this stuff under less-than-ideal conditions for a considerable period of time…
7. Store each white cell lysate in a 2 ml screw-top tube. DNA should be stable in this form for several weeks at room temperature, although refrigeration is recommended just to be safe.
8. To perform final extraction, add 150 µl of saturated NaCl (~ 6M NaCl) to 400 µl of white cell lysate.
9. Vortex and invert to mix; place on ice for 10′.
12. Centrifuge 5′ @ 12,000 RPM.
12. Add the supernatant to a 1.5 ml tube (550 µl); add 1000 µl absolute (100%) ethanol. Mix by inversion – the DNA precipitate should be visible at this point.
13. Spin 5′ @ 12,000 RPM; discard supernatant.
14. Wash w/ 1 ml 70% ethanol; air dry and resuspend in 100 µl of dilute TE (1:4 w/ H2O). Leave overnight at 4°C to resuspend completely. Check concentration and dilute to 100 ng/µl for stock. Freeze white cell lysates at -70°C for long-term storage.
Recommended field equipment for DNA extraction
1. Clinical centrifuge capable of at least 1,000 RPM, or hand-cranked model
2. 10 ml pipettes (minimum of 1 per extraction session – e.g. 1/day)
3. 15 ml Falcon tubes (1 per sample)
4. P-1000 Pipetteman
5. 1000 µl pipette tips (minimum of 1/sample)
6. 2 ml screw-top tubes
7. Gloves
8. Tube racks (for Eppendorfs and 15 ml tubes)
9. Blood collecting supplies (Vacutainers, needles [21 gauge if possible], plastic housings, tourniquets, alcohol preps, cotton, Band-Aids, biohazard bin for needles)
10. Ice chest (pack the other supplies inside for travel – and bring documentation for customs in case you are asked about all of those needles, white powders and solutions inside)

Inmproving Infection Efficiency Via Virus Centrifugation

1. Ultracentrifuge the virus at 50,000 x g for 90 min at 4°C.
Remove the supernatant. Drain carefully and well (preferably with a pipet) since the viral pellet is glassy and will be a filmy smear on the side of the tube.
 
2. Resuspend the virus to 0.5–1% of the original volume in TNE and incubate overnight at 4oC. Swirling during incubation may damage the virus. Gently pipet the solution to mix only after the overnight incubation, to allow diffusion of the virus. TNE is Tris buffer with NaCl and EDTA, which helps maintain the pH and is appropriate for storage if desired. Media can be used if the virus will be used immediately.
 
3. If desired, perform a second round of ultracentrifugation (Steps 1–2) by pooling previously concentrated virus.
 
4. This step is necessary only if injecting into animals, not if infecting cells in culture: Remove cellular debris and aggregated virus by low speed centrifugation (500 x g) for 5 min at 4oC.
 
5. Determine the viral titers of pre- and post-concentrated viral supernatants.
 
6. Infect target cells according to the Retroviral Gene Transfer and Expression

Immunofluorescence Double Staining Protocol Parallel Approach

1. Preparation of Slides

 

 A. Cell Lines

  • Grow cultured cells on sterile glass cover slips or slides overnight at 37 º C

  •  Wash briefly with PBS
  • Fix as desired. Possible procedures include:

    10 minutes with 10% formalin in PBS (keep wet)

    5 minutes with ice cold methanol, allow to air dry

    5 minutes with ice cold acetone, allow to air dry

  • Wash in PBS

 B. FrozenSections

 

  • Snap frozen fresh tissues in liquid nitrogen or isopentane pre-cooled in liquid nitrogen, embedded in OCT compound in cryomolds. Store frozen blocks at – 80 ºC.

  • Cut 4-8 um thick cryostat sections and mount on superfrost plus slides or gelatin coated slides. Store slides at – 80 ºC until needed.
  •  Before staining, warm slides at room temperature for 30 minutes and fix in ice cold acetone for 5 minutes. Air dry for 30 minutes.
  •  Wash in PBS

 C. Paraffin Sections

  • Deparaffinize sections in xylene, 2x5min.

  • Hydrate with 100% ethanol, 2x3min.
  •  Hydrate with 95% ethanol, 1min.
  •  Rinse in distilled water.
  •  Follow procedure for pretreatment as required.

2. Pretreatments of Tissue Sections

 

Antigenic determinants masked by formalin-fixation and paraffin-embedding often may be exposed by epitope umasking, enzymatic digestion or saponin, etc. Do not use this pretreatment with frozen sections or cultured cells that are not paraffin-embedded.

 

3. Procedure

 

Note: prior to perform double labeling, it is important to test each primary antibody individually and select the best pretreatment(s) for each antibody. It will be ideal if the two primary antibodies require same pretreatment. Otherwise, one should do a further test by treating sections with both pretreatments and then immunostain for each antibody individually. If both antibodies survive the “double pretreatments”, you are ready for immunohistochemistry double staining. Another alternative is to do pretreatments separately for each antibody staining.

  1.  Rinse Sections in PBS-Tween 20 for 2×2 min.

  2. Serum Blocking: incubate sections in normal serum blocking solution – species same as secondary antibody (for example: primary antibodies are mouse and rabbit, and secondary antibodies are horse anti-mouse, and goat anti-rabbit, so horse and goat serum block should be used).
  3. Primary Antibodies: incubate sections in the mixture of two primary antibodies (mouse and rabbit) at appropriate dilution in antibody dilution buffer for 1 hour at room temperature.
  4. Rinse in PBS-Tween 20 for 3×2 min.
  5. Secondary Antibodies: incubate sections in the mixture of two fluorescent conjugated secondary antibodies (FITC conjugated Horse anti-Mouse and Texas Red conjugated Goat anti-Rabbit) in PBS for 30 minutes at room temperature).
  6. Rinse in PBS-Tween 20 for 3×2 min.
  7. Counterstain with DAPI if desired for 20 minutes at room temperature.
  8. Rinse in PBS-Tween 20 for 3×2 min.
  9. Coverslip with anti-fade fluorescent mounting medium and seal with nail polish.
  10. Store slides in dark at 4 °C.

 4. Results:

  •  1st Primary Antibody Staining Sites ——————- green
  •  2nd Primary Antibody Staining Sites——————- red  
  • Double Staining Sites ———————————– yellow
  • Counterstained Nuclei ———————————- light blue