Cell Biology

Making Metaphase Spread

METAPHASE SPREAD:??
11/2/04

Materials:

Colcemid (GIBCO: cat #: 15210-040)
Hypotonic solution (pre-warmed 37C):?? 1:1???????? 0.4% KCl?? +?? 0.4% Sodium citrate
Fixative:?? 3:1???? MeOH + Acetic acid (e.g., 15 mL MeOH + 5 mL Acetic acid)
Pasteur transpipet

Procedure:

Grow and harvest cells:

  1. Grow cells to 85% confluence
  2. Add colcemid to cell culture at 10 ??l/mL
  3. Incubate for 2 hours (depends on cell cycle: longer cycle = longer treatment; H9 = 30 min.)
  4. Remove and collect growth medium and rinse cells with Hank’s or PBS
  5. Add 2 mL trypsin and reincubate the cells at 37??C, 5-7 min
  6. Re-add the collected medium from step 4 to stop the trypsin and resuspend cells
  7. Centrifuge the cells down at 1000 RMP, 6 min
  8. Aspirate the medium but leave small amount of fluid
  9. Flick the tube with finger to fully resuspend the cells

Addition of hypotonic solution:

  1. Add 5 drops of pre-warmed hypotonic solution slowly against the side of the tube
  2. Flick the tube with fingers until 1 mL had been added
  3. Bring the volume to about 2 mL with hypotonic solution
  4. Incubate at 37??C, 7min
  5. Centrifuge the cells down at 1000 RMP, 6 min
  6. Aspirate the medium but leave small amount to resuspend the cells

Fixation:

  1. Add 5 drops of fixative slowly against the side of the tube
  2. Bring the volume to 2 mL with fixative, can vortex if see clumps
  3. “Reverse bubble” to fully mix the cells
  4. Fix the cells at RT, 30 min
  5. Centrifuge, aspirate, and resuspend with finger as before
  6. Remove the clumps (if any) by vacuum from the side of tube
  7. Add fixative to 2 mL
  8. “Reverse bubble” and let stand at RT, 20 min
  9. Centrifuge, aspirate, and resuspend with finger as before
  10. Add fixative to 2 mL
  11. Cells are now ready to be dropped

Slide prepration (optional):

  1. Rinse slide with ice-cold water
  2. Rinse slide with fixative
  3. Drop cells flat on slide (optimal humidity: 50%-60%)
  4. Flood slide with fixative
  5. Dry slide on wet paper towels

Drop the cells onto pre-cleaned slide or treated slide above, rinse with fixative (optional), dry on wet tower papers, and observe for metaphase spreads with a phase-constrast microscope.

Bromodeoxyuridine Brdu Protocol

Bromodeoxyuridine uses nucleotide substitution to replace thymidine with uridine in DNA structure of dividing cells.??BrdU has been utilised ??in a number of in vitro and in vivo studies to label a variety of cellular sources from human olfactory epithelium ??and neural progenitors ??to neural stem cells.

Identification of the labelled cells can be applied both in vitro and in vivo to produce a nuclear staining pattern.

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BrdU protocol

Bromodeoxyuridine (BrdU) uses nucleotide substitution to replace thymidine with uridine in the DNA structure of dividing cells both in vitro and in vivo (Gage, 2000). ??BrdU has been utilised ??in a number of in vitro and in vivo studies to label a variety of cellular sources from human olfactory epithelium (Hahn et al., 2005) and neural progenitors (Nieoullon et al., 2005) to neural stem cells (Chu et al., 2004).

BrdU antibody from Abcam

Identification of the labelled cells can be applied both in vitro and in vivo to produce a nuclear staining pattern.

Protocol

Tissue and cells are ??fixed with 4% paraformaldehyde in vitro for 30 mins at 4??C, in vivo post fix for two hours at room temperature (r.t.)

Following fixation, wash in 0.1M Phosphate Buffered Saline (PBS) (pH 7.4) with 1% TritonX100 (3x 5 mins)

Incubate in HCl (1N) for 10 mins on ice to break open the DNA structure of the labelled cells

This is followed by HCl (2N) for 10 mins at r.t before moving them to an incubator for 20 mins at 37??C

Immediately after the acid washes, Borate buffer (0.1M) is added to buffer the cells for 12 mins at r.t.

Samples are then washed in wash in 0.1M PBS (pH 7.4) with 1% TritonX100 (3x 5 mins) at r.t.

Incubate in 0.1M PBS (pH 7.4) + 1% TritonX100 + Glycine (1M) + 5% normal goat serum (1hr) prior to incubating overnight (r.t) with anti-BrdU or a combination of anti-BrdU and other antibodies.

Following the incubation overnight wash in 0.1M PBS (pH 7.4) with 1% TritonX100 (3x 5min)

Samples can then be treated with a variety of secondary antibodies to visualise the anti-BrdU labelled cells (Fig 1 and Fig 2) including HRP conjugated secondaries with diaminobenzidine(DAB) or fluorescent conjugated antibodies such as AlexaFlour 488 or 533.

Tissue processing tips

In vivo and in vitro samples can be processed in this manner:

Gelatin embedded tissue (cut between 30-50??m)

OCT embedded tissue (cut between 10-30??m)

Tissue culture cells

NB: The acid steps haven???t shown any adverse affect on the labelling of any other antigens tested so far in double labelling. Incubation times haven???t needed to be altered with thickness of section and everything is treated in the same manner.

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BrdU Protocol InVitro??

Fig. 1 BRDU??

??In vitro dividing cells can be labelled with BrdU and following the protocol were visualised with HRP conjugated secondary antibodies and DAB to produce a nuclear staining pattern. ??Scale = 100??m

BrdU Protocol In Vivo

Fig. 2 BRDU

In vivo cells can be labelled either by injecting BrdU or alternatively label the cells prior to transplantation and then identify them using the protocol. ??Neural stem cells were labelled 48hrs prior to transplantation into a spinal cord injury at cervical level 4 the cells were identified throughout the dorsal ventral axis of the cord and had also begun to migrate rostro caudally from injection site. ??They were visualised here using the protocol followed by a TRITC conjugated secondary antibody Scale = 500??m.

BrdU

Fig. 3 BRDU

??In vivo cells were labelled prior to transplantation and then identified using Abcam Polyclonal anti-BrdU with a TRITC conjugated secondary antibody. Scale = 100??m

Antigen Specific Tcell Elispot

STEP 1:- PREWETTING WITH 70% ETHANOL FOR EACH WELL 50ul KEEP FOR 5 MIN.
STEP 2:-WASH WITH PBS 250ul FOR 3 TIMES.
STEP 3:- 100ul CAPTURE ANTIBODY 5ug/ml,(5ul/ml).STOCK CONC.1mg/ml.
STEP 4:- OVERNIGHT INCUBATION AT 4 DEGREE.APPROX .18 HRS.
WASH WITH PBS FOR 6 TIMES (300 ul/well).
STEP 5:- BLOCKING WITH FBS FOR EACH WELL 250ul FILTER WITH( .45 MILIPORE FILTER).
STEP 6:- 4 HRS INCUBATION.
INBETWEEN PREPARE EFFECTOR CELLS IN RPMI MEDIUM.
STEP 7:- SEED CELLS INTO WELL WITH 1 MILLION CELLS /ml .(100ul PER WELL).
FOR POSITIVE CONTROL
STEP 8:-ADD MITOGEN—(PHA) 50ul PER WELL.(+1)
STEP 9 :- OR ANTIGEN 50 ul PER WELL.{(-2)(-) control (-3)}
STEP 10:-INCUBATE FOR 18-24HRS IN 37 DEGREE CO2 INCUBATOR.
STEP 11:- WASH WITH 2 TIMES PBS AND 4 TIMES PBST APPROX 250-300ul.
ADD SECONDARY ANTIBODY(BIOTINYLATED).
20ul SEC ANTIBODY + 10 ml 7.5 % BSA in DPBS.???ADD 100UL PER WELL.
STEP 12:-INCUBATE FOR A ATLEAST 3HRS AT ROOM TEMP.
STEP 13:- DISCARD CONTENT OF PLATE..
STEP 14:- PREPARE BEFORE HALF HOUR 10 Ml DPBS IN PETRIDISH + 1 DROP A+ B REAGENT OF VICTASTAIN + 10 Ul T-20
STEP 15:- WASH 2 TIMES PBS & 4 TIMES WITH PBST
STEP 17:- ADD 100ul PER WELL & INCUBATE FOR 1 HR AT ROOM TEMP.
PREPARATION OF SUBSTRATE :-
A:- 2.5 ml OF DMF TAKEN IN 5 ml TUBE COVER WITH ALMINIUM FOIL + 1 TABLET OF AEC.
B:- 47.5 ml DW SIGMA TAKEN IN 50 ml TUBE + 280 ul SODIUM ACETATE + 180 ul ACETIC ACID
STEP 18:- MIX A+ B and ADD 25ul H202.
STEP 19:-10 ML MIXTURE WAS FILTERD BY 0.45 ul.
STEP 20:-AVIDIN ???ENZYME COMPLEX DISCARD .
STEP 21:-WAS PLATE WITH 3 TIMES PBST THEN 3 TIMES WITH PBS.
STEP 22:-100ul/well AEC SUSTRATE ADDED.
STEP 23:-INCUBATE FOR 5 MIN IN DARK.
STEP 24:-STOP DEVELOPMENT UNDER TAP WATER.
STEP 25:-DRY WELL WITH PAPER TOWEL.
STEP 26 :- OVERNITE DRY PLATE IN DARKNESS.

Culture And Maintenance Of Human Embryonic Stem Cells

Culture and Maintenance of Human Embryonic Stem Cells

Human Embryonic Stem Cell Protocols

HUMAN EMBRYONIC STEM CELL PROTOCOLS

Apoptosis Detection Using Terminal Transferase And Biotin16Dutp Tunel Enzyme Method

Description:??This protocol is used for detection and quantification of apoptosis (programmed cell death) at single cell level, based on labeling of DNA strand breaks (TUNEL technology). Cleavage of genomic DNA during apoptosis may yield double stranded as well as single strand breaks (“nicks”), which can be identified by labeling free 3’-OH terminal with modified nucleotides in an enzymatic reaction.

Positive Controls:??1) Incubate sections with DNase I (3000U/ml in 50 mM Tris-HCl, pH 7.5, 1mg/ml BSA) for 10 minutes at 15-25 ??C to induce DNA strand breaks, prior to labeling procedure. 2) using known positive control is an alternative.

Negative Control:??incubate sections with label solution only (without terminal transferase) instead of TUNEL reaction mixture.

Solutions and Reagents:

TdT Buffer Stock Solution (125mM Tris-HCl, 1M Sodium Cacodylate, 1.25mg/ml BSA, pH 6.6):??

Tris-HCl (MW 157.6) ———————————– 1.97 g

Sodium cacodylate, Trihydrate (MW 214.0) ——— 21.4 g

BSA ——————————————————- 0.125 g

Distilled water —————————————— 100 ml

Adjust pH to 6.6 and aliquot and store at ???20????C.

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Cobalt Chloride Stock Solution (25mM Cobalt Chloride in Distilled Water):

??

Cobalt chloride, Hexahydrate (MW 237.9) ———— 0.6 g

Distilled water ——————————————- 100 ml

Mix to dissolve. Aliquot and store at ???20????C.

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TdT Reaction Buffer (25mM Tris-HCl, 200 mM Sodium Cacodylate, 0.25 mg/ml BSA, 1mM Cobalt Chloride):

??

TdT Buffer Stock Solution —————————— 40 ul

Cobalt Chloride Stock Solution ————————- 8 ul

Distilled water ——————————————- 160 ul

Mix well. Store at?????20 ??C

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TdT Storage Buffer (60mM K-phosphate, pH 7.2, 150mM KCl, 1mM 2-Mercaptoethanol, 0.5% Triton X-100, 50% glycerol)

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?????????? To make the buffer:

?????????? K2HPO4 (MW174.18) ———————————— 1.05 g

?????????? KCl (FW 74.55) ——————————————- 1.12 g

?????????? Distilled water ——————————————– 50 ml

????????????Stir to dissolve and adjust pH 7.2 using concentrated HCl. Add 50 ml of glycerin (100% glycerol), 0.5 ml of Triton X-100, and 8 ul of 2-Mercaptoethanol (99% Solution. FW 78.13). Store at ???20 ??C

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Enzyme Reagent:

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????????????Terminal Transferase (TdT) (Roche Diagnostic) —— 4 ul

????????????TdT Storage Buffer ————————————– 100 ul

????????????Mix well and store at ???20 ??C

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Label Reagent:

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????????????Biotin-16-dUTP (Roche Diagnostic) ——————— 4 ul

????????????TdT Reaction Buffer ————————————– 1 ml

????????????Mix well and store at ???20 ??C

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TdT Reaction Mixture:

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??????????????Enzyme Reagent —————————————– 100 ul

??????????????Label Reagent ——————————————– 900 ul

??????????????Mix just before use. The remaining 100 ul of Label Solution can be used for negative control.

????????????????????????
Stop Wash Buffer (300mM NaCl, 30mM Sodium Citrate):

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NaCl (MW 58.44) —————————————– 1.75 g

Sodium citrate, Trihydrate (MW294.11) ————— 0.88 g

Distilled water ——————————————– 100 ml

Mix to dissolve and store at room temperature.

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Procedure:

  1. Deparaffinize??sections in 2 changes of xylene for 5 minutes each, and hydrate with 2 changes of 100% ethanol for 3 minutes each, and 95% ethanol for 1 minute.

  2. Rinse in distilled water.
  3. Pretreatment:??Use proteinase K digestion method. Note: for frozen sections or culture cells grown on slides, incubate with 0.2% Triton X-100 in PBS-Tween for 30 minutes is required.
  4. Rinse sections in 2 changes of PBS-Tween 20, 2 minutes each.
  5. Peroxidase Blocking: incubate sections in 3% H2O2 in PBS for 10 minutes to block endogenous peroxidase activity.
  6. Rinse in PBS-Tween 20 for 3×2 min.
  7. Pre-incubation:??incubate sections in TdT Reaction Buffer for 10 minutes.
  8. TdT Reaction: incubate sections in TdT Reaction Mixture for 1-2 hours at 37-40????C in humidified chamber.
  9. Stop Reaction:??rinse sections in stop wash buffer for 10 minutes.
  10. Rinse in??PBS-Tween 20??for 3x2min.
  11. Detection:??incubate sections with Streptavidin-HRP in PBS for 20 minutes at room temperature.
  12. Rinse in??PBS-Tween 20??for 3x2min.
  13. Chromagen/Substrate:??incubate sections with DAB for 1-2 minutes.
  14. Rinse in tap water.
  15. Counterstain??with Gill’s hematoxylin for 30 seconds.
  16. Rinse in running tap water for 5 minutes.
  17. Dehydrate through 95% ethanol for 5min, 100% ethanol for 2x3min.
  18. Clear??in xylene for 2x5min.
  19. Coverslip??with xylene based mounting medium.

Apoptosis Detection Using Apoptag Peroxidase In Situ Apoptosis Detection Kit From Chemicon S7100

Description:??ApopTag????In Situ Apoptosis Detection Kits label apoptotic cells in research samples by modifying DNA fragments utilizing terminal deoxynucleotidyl transferase (TdT) for detection of apoptotic cells by specific staining. The Kit has been qualified for use in histochemical and cytochemical staining of the following specimens: formalin-fixed, paraffin-embedded tissues, cryostat sections, cell suspensions, cytospins, and cell cultures. Whole mount-methods have been developed.

Principles of the Procedure:??The reagents provided in ApopTag????Peroxidase Kits are designed to label the free 3’OH DNA termini in situ with chemically labeled and unlabeled nucleotides. The nucleotides contained in the Reaction Buffer are enzymatically added to the DNA by terminal deoxynucleotidyl transferase (TdT). TdT catalyzes a template-independent addition of nucleotide triphosphates to the 3′-OH ends of double-stranded or single-stranded DNA. The incorporated nucleotides form an oligomer composed of digoxigenin-conjugated nucleotide and unlabeled nucleotide in a random sequence. The ratio of labeled to unlabeled nucleotide in ApopTag????Peroxidase Kits is optimized to promote anti-digoxigenin antibody binding. The exact length of the oligomer added has not been measured.??

DNA fragments which have been labeled with the digoxigenin-nucleotide are then allowed to bind an anti-digoxigenin antibody that is conjugated to a peroxidase reporter molecule. The bound peroxidase antibody conjugate enzymatically generates a permanent, intense, localized stain from chromogenic substrates, providing sensitive detection in immunohistochemistry or immunocytochemistry (i.e. on tissue or cells). This mixed molecular biological-histochemical systems allows for sensitive and specific staining of very high concentrations of 3′-OH ends that are localized in apoptotic bodies.??

The ApopTag????system differs significantly from previously described in situ labeling techniques for apoptosis, in which avidin binding to cellular biotin can be a source of error. The digoxigenin/anti-digoxigenin system has been found to be equally sensitive to avidin/biotin systems. The sole natural source of digoxigenin is the digitalis plant. Immunochemically-similar ligands for binding of the anti-digoxigenin antibody are generally insignificant in animal tissues, ensuring low background staining. Affinity purified sheep polyclonal antibody is the specific anti-digoxigenin reagent used in ApopTag????Kits. This antibody exhibits <1% cross-reactivity with the major vertebrate steroids. In addition, the Fc portion of this antibody has been removed by proteolytic digestion to eliminate any non-specific adsorption to cellular Fc receptors.

Solutions and Reagents:

  • Equilibration Buffer: 3.0 mL -15??C to -25??C
  • Reaction Buffer 2.0 mL -15??C to -25??C
  • TdT Enzyme 0.64 mL -15??C to -25??C
  • Stop/Wash Buffer 20 mL -15??C to -25??C
  • Anti-Digoxigenin-Peroxidase* 3.0 mL 2??C to 8??C
  • Plastic Coverslips 100 ea. Room Temp.
  • Note: Separate purchase of DAB (Peroxidase Substrate) is required. It is not supplied with this kit.
  • Number of samples per kit: Sufficient materials are provided to stain 40 tissue specimens of approximately 5 cm2??each when used according to instructions. Reaction Buffer will be fully consumed before other reagents when kits are used for slide-mounted specimens.

Procedure:

  1. Deparaffinize??sections in 2 changes of xylene for 5 minutes each, and hydrate with 2 changes of 100% ethanol for 3 minutes each, and 95% ethanol for 1 minute.

  2. Rinse in distilled water.
  3. Pretreatment:??Use??proteinase K digestion method. Note: for frozen sections or culture cells grown on slides, incubate with 0.2% Triton X-100 in PBS-Tween for 30 minutes is required.
  4. Rinse sections in 2 changes of PBS-Tween 20, 2 minutes each.
  5. Peroxidase Blocking: incubate sections in 3% H2O2 in PBS for 10 minutes to block endogenous peroxidase activity (for frozen section, use 0.3% H2O2 in methanol)
  6. Rinse in PBS-Tween 20 for 3×2 min.
  7. Pre-incubation:??incubate sections in Equilibration Buffer for 5-10 minutes.
  8. TdT Reaction: incubate sections in Working Strength TdT Enzyme (Mixture of 100 ul Reaction Buffer and 30 ul TdT Enzyme) for 1 hours at 37??C in humidified chamber.
  9. Stop Reaction:??rinse sections in Working Strength Stop/Wash Buffer for 10 minutes.
  10. Rinse in??PBS-Tween 20??for 3x2min.
  11. Detection:??incubate sections with Anti-Digoxigenin Conjugate (Peroxidase) for 30 minutes at room temperature.
  12. Rinse in??PBS-Tween 20??for 3x2min.
  13. Chromagen/Substrate:??incubate sections with DAB for 1-2 minutes.
  14. Rinse in tap water.
  15. Counterstain??with Gill’s hematoxylin for 30 seconds.
  16. Rinse in running tap water for 5 minutes.
  17. Dehydrate through 95% ethanol for 5min, 100% ethanol for 2x3min.
  18. Clear??in xylene for 2x5min.
  19. Coverslip??with xylene based mounting medium.

Results:

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Staining Pattern: Nuclear??(Search Images)

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References:

  1. Gavrieli Y, et al (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 119:493-501.

  2. ??Charriaut-Marlangue C and Ben-Ari Y (1995) A cautionary note on the use of TUNEL stain to determine apoptosis. NeuroReport 7:61-64.

  3. Ay I, et al (2001) Intravenous basic fibroblast growth factor (bFGF) decreases DNA fragmentation and prevents downregulation of Bcl-2 expression in the ischemic brain following middle cerebral artery occlusion in rats. Molecular Brain Res. 87:71-80.

Tem Immunogold Labeling Protocol Preembedding Method

Procedure:

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1.??????Sectioning:??Vibratome sections at 50 um thick.

2.??????Collect sections in 0.1M PB.

3.??????Pretreatment:??treat sections with 1% sodium borohydride solution for 30 minutes.

4.??????Rinse many times in 0.1M PB to remove bubbles.

5.??????Rinse in PBS for 3x5min

6.??????Serum Blocking:??incubate in blocking buffer for 30 minutes.

7.??????Primary Antibody:??incubate sections with primary antibody (appropriate diluted in blocking buffer) overnight at room temperature.

8.??????Rinse in PBS for 3x5min.

9.??????Washing Buffer:??incubate in washing buffer for 30 minutes.

10.??Secondary Antibody:??incubate sections in gold-conjugated secondary antibody diluted 1:50 in washing buffer for 3 hours.

11.??Rinse 4×5 min in washing buffer and then 4×5 min in PBS.

12.??Post-fixation1:??fix sections with 2% glutaraldehyde in PBS for 10 minutes.

13.??Rinse 4×5 minutes in PBS.

14.??Silver enhancement:

????????????Rinse in 0.2M citrate buffer 3x2min.

????????????Incubate in silver enhancement solution for 3-9 minutes for EM and 6-12 minutes for LM (If room temperature is very low, a longer time may be needed such as 20-30 min).

????????????Rinse in 0.2M citrate buffer 2x5min

????????????Rinse in 0.1 M PB 4×5 min

15.??Post-fixation2:??fix sections with 1% OsO4 in 0.1M PB for 1 hour.

16.??Rinse in 0.1M PB for 3x5min.

17.??Dehydration:??dehydrate in 50%, 70% and 95% ethanol for 10 minutes each.

18.??100% ethanol 2×15 min.

19.??Propylene oxide 2×15 min.

20.??Incubate sections in 1:1 mixture of Embed-812 & propylene oxide for overnight.

21.??Transfer sections to straight Embed-812 for 2 hours.

22.??Embedding:??flat-embedding, and bake in 62 C oven for 48 hours.

23.??Sectioning:??choose the region of interest, cut off and glue on blank flat-end beam capsule using super glue. Bake to dry for 30 minutes to 1 hour. Trim and cut ultrathin sections at 60-90 nm.

24.??Contrast Staining:??stain sections with uranyl acetate for 15 minutes and lead citrate for 5 minutes.

27.??Observation.

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Solutions and Reagents:

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0.2M Phosphate Buffer (0.2M PB):

To prepare 1 liter,

Na2HPO4,??——————– 21.8 g

NaH2PO4??——————— 6.4 g

Distilled water ————- 1000 ml

Mix to dissolve and adjust pH to 7.4

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0.1M Phosphate Buffer (0.1M PB):

To prepare 1 liter,

0.2M PB ——————— 500 ml

Distilled water ————– 500 ml

10X Phosphate Buffered Saline (PBS):

To prepare 1 liter,

Na2HPO4??——————— 10.9 g

NaH2PO4??——————— 3.2 g

NaCl ————————— 90 g

Distilled water ————– 1000 ml

Mix to dissolve and adjust pH to 7.4

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0.2M Citric Acid:

To prepare 100 ml,

Citric acid (monohydrate) —— 4.2 g

Distilled water ——————– 100 ml

0.2M Sodium Citrate Buffer (fresh):

To prepare 100 ml,

Sodium citrate (dihydrate) —— 5.88 g

Distilled water ——————— 100 ml

Adjust pH to 7.4 with 0.2M citric acid (keeps refrigerated).

Fixative (4% Paraformaldehyde and 0.5% Glutaraldehyde in 0.1M PB):

To prepare 1 liter,

Paraformaldedyde ————– 40 g

0.1M PB ————————— 1000 ml

Heat to 65 C. Add a few drops of 1N NaOH to clear the solution. Stir to dissolve. Filter the solution and add 10 ml of 50% glutaraldehyde.

Post-fixative (1% Osmium Tetroxide in 0.1M PB):

To prepare 20 ml,

4% osmium tetroxide ———- 5 ml

0.2M PB ————————— 5 ml

0.1M PB ————————– 10 ml

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2% Glutaraldehyde in PBS:

To prepare 100 ml,

50% glutaraldyhyde ———— 4 ml

PBS ——————————– 100 ml

1% Sodium Borohydrite Solution in 0.1M PB:

To prepare 100 ml,

Sodium borohydrite ————– 1 g

0.1M PB —————————- 100 ml

Stir to dissolve.

Blocking Buffer (1% BSA, 3% NGS, 0.04% Triton in PBS):

To prepare 100 ml,

Triton X-100 ———————– 0.04 ml

BSA ———————————- 1 g

Normal goat serum ————– 3 ml

PBS ———————————- 100 ml

Stir to dissolve.

Washing Buffer (0.8% BSA, 0.1% Fish Gelatin in PBS).

To prepare 100 ml,

BSA ———————————- 0.8 g

Fish gelatin ———————— 0.1 ml

PBS ——————————— 100 ml

Secondary Antibody:

1nm gold conjugated secondary antibody from Amersham. Dilute the antibody 1:50 in washing buffer.

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Silver Enhancement Solution:

Kit from Amersham and prepare solution according to the instruction provided.

Embed-812:

Embed-812 Kit from EMS containing Embed-812, DDSA, NMA, and DMP-30. Make Embed-812 according to instruction provided with the kit.

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1:1 Mixture of Embed-812 & Propylene Oxide:

To prepare 20 ml,

Embed-812 ————————- 10 ml

Propylene oxide ——————- 10 ml

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5% Uranyl Acetate Solution:

To prepare 50 ml,

Uranyl acetate ——————— 2.5 g

Distilled water ——————— 50 ml

Cover with foil and stir overnight.

Add 10 drops of glacial acetic acid and Store solution in 4 C for 3 month.

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Reynold???s Lead Citrate Solution:

To prepare 50 ml, add chemicals in distilled water in following order

Lead nitrate ——————————- 1.33 g

Sodium citrate, dihydrate ————— 1.76 g (solution becomes cloudy when sodium citrate is added)

1N NaOH ———————————– 5 ml????(solution becomes clear when NaOH is added)

Distilled water —————————– 30 ml

Stir for 10 minutes to dissolve and add additional 15 ml of distilled water. Store solution for 3-6 months at 4 C. Note: the mount of NaOH is very important. Too much will make solution cloudy.

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Epon 812:

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Final Volume

EM bed-812

DDSA

NMA

DMP-30 (2%)

20.91 ml

10 ml

4.5 ml

6 ml

0.41 ml

31.37 ml

15 ml

6.75 ml

9 ml

0.62 ml

41.82 ml

20 ml

9 ml

12 ml

0.82 ml

52.28 ml

25 ml

11.25 ml

15 ml

1.03 ml

????????????????Mix all four components in plastic beaker and stir with wood stick.

Toluidine Blue Staining Protocol For Plastic Sections Or Electron Microscopy Tem Thick Semithin

Description:??This method is used for staining of TEM thick sections. The stained sections can be used as guideline to determine the area of interest and further trimming of Embed blocks. Therefore precise ultra-thin sections can be cut and mounted on TEM grids. This stain can also be used for staining of plastic sections of small tissue samples such as peripheral nerve, sciatic nerve, etc. for general morphological analysis.

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Fixation:??4% formaldehyde and 1% glutaraldehyde in phosphate buffer.

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Reagents:

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1% Toluidine Blue and 2% Borate in Distilled Water

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???? Toluidine Blue O —————————- 1 g

???? Sodium Borate (Borax) ——————— 2 g

???? Distilled Water —————————— 100 ml

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Dissolve the sodium borate in the water, then add the toluidine blue powder and stir until dissolved. Filter the stain solution (use syringe filter) before use (Note: The borax makes the stain alkaline so it will help penetrating to the epoxy sections).

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Procedure:

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1. Cut thick (semithin) sections at 0.5 um or 1.0 um.

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2.??Use a metal loop to collect thick sections, and transfer sections to a drop of distilled water on a glass slide.

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3. Dry sections down on a glass slide by placing the slide on a slide warmer or 40 wt lamp.

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4. After the sections are completely dried, cover with a few drops of staining solution (with the heat source still on) for 1-2 minutes depending on the darkness of staining you would like to achieve.

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5. Rinse off excess stain gently with distilled water.

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6. Air dry the slide.

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7. Coverslip with regular mounting medium.

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Results:

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Cells and nuclei stained blue.