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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
- 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
- Paper stapler
- 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
- 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
- Tape the resulting plastic tube once, at one third the distance from one extremity. Make a continuous ring of tape for maximum strength
- 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
- 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
- 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
- Gently spread the cut and introduce the young inflorescence. As secondary inflorescences are produced guide them in the collector or remove them
- 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
Inmproving Infection Efficiency Via Virus Centrifugation
Immunofluorescence Double Staining Protocol Parallel Approach
1. Preparation of Slides
A. Cell Lines
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Grow cultured cells on sterile glass cover slips or slides overnight at 37 º C
- Wash briefly with PBS
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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
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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.
-
Rinse Sections in PBS-Tween 20 for 2×2 min.
- 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).
- 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.
- Rinse in PBS-Tween 20 for 3×2 min.
- 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).
- Rinse in PBS-Tween 20 for 3×2 min.
- Counterstain with DAPI if desired for 20 minutes at room temperature.
- Rinse in PBS-Tween 20 for 3×2 min.
- Coverslip with anti-fade fluorescent mounting medium and seal with nail polish.
- 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
Protocol For Cryosectioning
While the timing of the various steps in this protocol are probably not critical, I tend to prefer to process the tissue all at once to ensure that RNA and/or proteins do not get degraded.
Solutions
20% Paraformaldehyde/4% Paraformaldehyde-PBS
200 g paraformaldehyde
1 ml 10N NaOH
up to 1 liter with Q, heat to 65° to dissolve, aliquote and store at -20°.
Mix 100 ml 20% Paraformaldehyde with 50 ml 10X PBS and bring up to 500 ml with Q
filter, and store at 4° for up to 2 weeks
Sucrose/PBS
30% sucrose 150 g sucrose
up to 500 ml with 1X PBS
filter sterilize and store at room temperature
Procedure
• Dissect and fix the tissue in fresh 4% paraformaldehyde on ice for 5-10 minutes.
• Wash for 5 minutes in 1X PBS and repeat.
• Transfer to 30% sucrose until the tissue sinks (5-10 minutes depending on the size of the tissue).
• Transfer through a 1:1 mixture of OCT:sucrose and then into OCT.
• Place the tissue in the cryomold, overlay with OCT, orient and freeze quickly on dry ice.
• Once the tissue is in the mold with OCT it should be oriented and frozen quickly because a film can form on the top of the mold (where the OCT is exposed to air) and make moving the tissue difficult.
• If the size of the mold is small enough place each block into an eppendorf tube and store at -80°C.
• Cut 20 micron sections and place on silinized superfrost slides (Protocol S.6).
• For best results, proceed immediately with immunohistochemical staining.