Histological Protocol
     
Fluorescent Label in Bone
     
 

The figure below contrasts the intensity of the GFP signal in a paraffin and standard frozen section and followed by the improvement in quality of frozen section when prepared with a tape transfer methodology (Cryojane ).

 
     
 

Protocol for Frozen section of GFP Bone:

    1. Fix the bone in 4% Paraformaldehyde at 4ºC under constant agitation for 3 days.
    2. Decalcification in 14% EDTA solution at 4ºC or RT under constant agitation for 3~5 days (change fresh 14% EDTA solution every 24 hours).
    3. Wash in PBS for 2 hours.
    4. Soak in 30% Sucrose in PBS at 4ºC under constant agitation overnight.
    5. Embed bone in OCT compound.
    6. Cut 5µm thick frozen sections using cryostat and allow the sections to dry ( keep in
      dark).
    7. Rinse in PBS 5min x 2.
    8. Soak in 1mM MgCl2 in PBS for 30min.
    9. Rinse in PBS 5min.
    10. Mount coverslide using 50% Glycerol in PBS.
    11. Examine by fluorescence microscopy.

Solution:

4% Paraformaldehyde in 0.1M PBS:
  • Heat PBS to ~ 65ºC to dissolve Paraformaldehyde,then cool it and adjust pH to 7.4. ( use fresh solution)

Decalcification solution:

  • 280 g EDTA
  • ddwater 1.5 L
  • 180 ml Ammonium Hydroxide
  • Mix well and adjust pH to 7.1 with Ammonium Hydroxide
  • Add water to 2 L.

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Embedding Bones for Frozen Section:

    1. Fill an ice bucket with dry ice pellets and place a 500ml beaker in the middle.
    2. Put approximately 200ml of 2-MethylButane in the beaker and cover the bucket. (The MethylButane will get extremely cold, allowing you to ‘flash freeze’ your samples).
    3. Label your molds and fill the molds with Frozen Embedding Medium (Thermo Shandon, Pittsburgh, PA).
    4. Immerse bone in embedding medium and adjust right position as needed.
    5. Test 2-MethylButane with dry ice pellets, if it does not boil, it is ready.
    6. Use a forceps to keep the embedding mold horizontaly for few seconds until the embedding medium is frozen in pre-cold Methybutane and allow it to sit to the bottom of the beaker for at least 2 minutes to ensure it is frozen all the way through.
    7. Frozen samples can be placed on dry ice in the bucket until all samples are done. Wrap your samples in aluminiumfoil.
    8. Place in a sealed container and store your samples at -20ºC or -80ºC.

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Trap Stain:

 Buffer I Solution:    
  Sodium Acetate Anhydrous (Sigma S-2889)
9.2g
  
  L- (+)Tartaric Acid (Sigma T-6521)
11.4g
  
  Distilled Water
950ml
  
  Glacial Acetic Acid
2.8ml
  
  • Dissolve and adjust pH to 4.7-5.0 with 5M Sodium Hydroxide
  • Bring total volume to 1L
  • (5M NaOH: Sodium Hydroxide Pellets 50g, Distilled Water—250ml)

Buffer II Solution:    
  Naphthol AS-BI Phosphate (Sigma N-2125) store at –20º
0.1g
  
  Ethylene Glycol Monoethyl Ether (Sigma E-2632)
5ml
  

 

 Buffer III Solution:    
  Sodium Nitrite (Sigma S-2252)
1g
  
  Distilled Water
20ml
  

 

 Buffer IV Solution:    
  Pararosaniline Chloride (Sigma P-3750)
1g
  
  2N HCL
20ml
  
  • Heat to 60º; C for 5 min do not boil.
  • Filter through kimwipe.
  • (2N HCL: HCL, 83ml; Distilled water, 417ml)

 

Staining Procedure:

  1. Preheat to 37°C 2 Coplin jars with 50ml of Buffer I Solution each.
  2. Take one Coplin jar and add 0.5 ml of Buffer II Solution, add slides and incubate at 37°C for 45 minus.
  3. A few min before the time is up, mix 1 ml of Buffer III Solution and 1 ml of Buffer IV solution for 30 sec and let it sit for 2 min.
  4. Add this mixed solution to the other Coplin jar of 50 ml Buffer I Solution, mix and add the slides without rinsing.
  5. Incubate at room temp ~5 min.
  6. Rinse, counterstain with hematoxylin for 40 sec and put slides in 0.05% Ammonia water.
  7. Dehydrate through alcohol, clear in xylene and mount.

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Alizarin Red S Staining - Calcium
   
Alizarin Red S Solution:
    
Alizarin Red S
2 gm
  
Distilled Water
100 ml
  
  • Mix the solution and adjust the pH to 4.1-4.3 using 0.5% ammonium hydroxide. The pH is critical - make fresh.
 
 
Acetone – Xylene:
    
Acetone
25 ml
  
Xylene
25 ml
  
Make fresh.

Procedure:

  1. Rinse slides rapidly with distilled water.
  2. Alizarin red S solution, 30 sec to 3 min, checking microscopically for the orange-red color.
  3. Shake off excess dye.
  4. Acetone:- 20 dips.
  5. Acetone-xylene:- 20 dips.
  6. Clear in xylene, mount in permount.

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Goldner Trichrome Staining
     
Weigert Hematoxyline:    
       
Solution A    
  Hematoxyline
5 g
  
  95% Alcohol
500 ml
  
       
Solution B
5 g
  
  Ferric chloride
5.8 gl
  
Deionized water
500 ml
  
  Concentrated Chlorhydric Acide
5.0 ml
  
  • 24 hours before use, mix solutions A and B in equal amounts
    Stable for 8 days.
 
 
Fushine-Ponceau:
    
  Fushine acid
0.167 g
  
  Ponceau S
0.667 g
  
  Deionized water
500 ml
  
  Acetic Acid (glacial)
1.0 ml
  
  • Shake well and filter.
 
 
Orange G:
    
  Phospho-molybdic Acid
25 g
  
  Deionized water (Shake well)
500 ml
  
  Add Orange G
10 g
  
  • Shake well and filter.
 
 
Light Green:
    
  Light green
1.5 g
  
  Deionized water
500 ml
  
  Acetic Acid (glacial)
1 ml
  
  • Shake well and filter.
  Acetic Acid 1%
 


Staining Procedure:

    1. Put slides in deionized water.
    2. Mordant in Bouin’s Fixative (Poly Scientific Inc. Catalog# s129-16oz) at room temperature overnight.
    3. Rinse with deionized water frequently for 10 minutes.
    4. Weight Hematoxyline 15 minutes.
    5. Rinse with deionized water frequently for 10 minutes.
    6. Fushine-ponceau 30 minutes.
    7. Rinse with 1% acetic acid rapidly.
    8. Orange G 8 minutes.
    9. Rinse with 1% acetic acid rapidly.
    10. Light Green 20 minutes
    11. Rinse with 1% acetic acid rapidly.
    12. Dehydrate through alcohol, clear in xylene and mount.

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Fluorescent Immunostaining for BrdU:

    1. .
       
      • Wash frozen sections with PBS 2x.
      • 0.1%Pepsin in 0.1N HCL in PBS, incubate sections at 37ºC for 50min.
      • Incubate with 2N HCL in distilled water 30 min at 37ºC.
      • Rinse with PBS 5x.
      • 5% normal donkey serum in PBS with 1% BSA at RT for 1 hour.
      • Mouse anti-BrdU (Singa) diluted to 1:1000 with 1% NDS, 1%BSA in PBS, incubate at 4ºC overnight.
      • Rinse with PBS 3x.
      • Donkey anti-Mouse IgG conjugated-Tritc 1:100 (Jackson ImmunoResearch Lab, Inc. Pennsylvania) in 1% NDS, 1%BSA in PBS, incubateat RT for 1 hour or overnight at4ºC.
      • Rinse with PBS 3x.
      • Mount with 50% glycerol in PBS

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Enzyme Immunostaining for BrdU:

    1. Wash frozen sections with PBS 2x.
    2. Put them in 0.1% Pepsin in 0.1N HCL (in PBS) at 37ºC for 50min.
    3. Then in 0.3% hydrogen peroxide in PBS, 30min. Then rinse with PBS 3x.
    4. Then in 5% normal goat serum in PBS with 1% BSA at RT for 1 hour.
    5. Mouse anti-BrdU diluted to 1:1000 with 1% NGS and1%BSA in PBS, incubateat 4ºC overnight.
    6. Rinse with PBS 3x.
    7. Goat Biotinylated anti-Mouse IgG 1:200 in 1% NGS, 1%BSA in PBS, incubate at RT for 1 hour. Rinse in PBS 3x.
    8. Incubate with ABC reagent in PBS containing 0.1% Tween 20 at RT for 1 hour.. (ABC reagent solution: 5ml PBS/0.1% Tween 20 with 1 drop A and 1 drop B, mix well).
    9. Rinse with PBS 3x.
    10. DAB reaction: 2.5ml of distilled water + 1 drop Buffer + 2 drop DAB solution + 1 drop hydrogen peroxide. ~Needs about 8 min to stop DAB reaction.
    11. Rinse with distilled water 3x. Counterstain and mount.

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Culture Cell Fluorescence Immunostaining for BrdU:

    1. Wash plates with PBS 3x.
    2. Fix them in 4%PFA, 20 min at RT.
    3. Wash them in PBS 3x.
    4. 0.5% Triton X-100 in PBS, 15min at 4ºC
    5. Wash slides in PBS 3x.
    6. 2N HCL( in Distilled water) 30min at 37ºC( DNA denaturation).
    7. Wash in PBS 5x
    8. Incubate slides with 1%BSA in PBS at RT for 30min.
    9. Mouse anti-BrdU diluted to 1:1000 with 1% NDS and1%BSA in PBS, incubate at RT for 2hrs.
    10. Wash with PBS 3x.
    11. 1: 100 dilution of Rhodamine(Tritc) conjugated Donkey anti mouse IgG with 1% NDS,1%BSA in PBS, incubate at RT for 1 hr.
    12. Wash in PBS 3x
    13. Mount with 50% glycerol in PBS.

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Culture Cell Enzyme Immunostaining for BrdU:

    1. Wash plates with in PBS 3x.
    2. Put 4%PFA 20 min at RT.
    3. Wash with PBS 3x.
    4. 0.5% Triton X-100 in PBS, 15min at 4ºC
    5. Wash with PBS 3x.
    6. Incubate with 2N HCL( in Distilled water) 30min at 37ºC( DNA
      denaturation).
    7. Wash with PBS 5x
    8. 1% BSA ,incubate for 30min at RT.
    9. Mouse anti-BrdU diluted to 1:1000 with 1% NGS,1%BSA in PBS,
      incubate at RT for 2hrs.
    10. Wash withPBS 3x.
    11. Dilute Goat Bionytilated anti-Mouse IgG to 1:200 with1% NGS and
      0.1% BSA in PBS, incubate at RT for 1 hour. Rinse with PBS 3x.
    12. ABC at RT for 1 hour. Rinse in PBS 3x.
      ( ABC solution: 5ml PBS with 1 drop A and 1 drop B, mix well)
    13. DAB reaction: 2.5ml of distilled water + 1 drop Buffer + 2 drop DAB
      solution+1 drop hydrogen peroxide. Needs about 8 min to stop DAB reaction.
    14. Rinse with distilled water 3x.
    15. Countstain with Mayer’s Hematoxylin

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Frozen Section- Enzyme Immunostaining for CD31 (endothelium):

    1. Rinse slides 3 times with PBS.
    2. Put them in 3% hydrogen peroxide in Distill water, 30min. Then rinse in PBS 3x.
    3. Incubate with 1xPower Block at RT for 20min.
    4. Rinse 3 times with PBS.
    5. Put them in Rat anti-Mouse CD31 diluted to 1:100 with 1% NRS in 0.1%BSA+ PBS, incubate at 4C overnight. Rinse in PBS x3.
    6. Then in Ribbit Bionytilated anti-Rat IgG 1:200 in PBS with 1% NRS and 0.1% BSA, incubate at RT for 1 hour. Rinse in PBS 3x.
    7. ABC at RT for 1 hour. Rinse in PBS 3x. ( ABC solution: 5ml PBS with 1 drop A and 1 drop B, mix well)
    8. DAB reaction: 2.5ml of distilled water + 1 drop Buffer + 2 drop DAB solution + 1 drop hydrogen peroxide. ~Needs about 8 min to stop DAB reaction.
    9. Rinse in distilled water 3x.
    10. Countstain with Mayer’s Hematoxylin.

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Immunostaining for Factor VIII Related Antigen (endothelium):

    1. Deparaffinize and rehydrate tissue sections. Rinse 3 times with PBS.
    2. 0.1%Pepsin in 0.1N HCL in PBS incubated sections at 37°C for 40min.
    3. Rinse with PSA 3x
    4. 0.3% hydrogen peroxide in PBS, 30min. Then rinse in PBS 3x
    5. 5% normal goad serum in PBS at RT for 1 hour.
    6. Rabbit anti-Human Factor VIII Related Antigen diluted to 1:500 with 1% NGS in PBS, incubated at 4°C overnight. Rinse in PBS x3.
    7. Goat Bionitylated anti-Rabbit IgG 1:200 in 1% NGS in PBS, incubated at RT for 1 hour. Rinse in PBS 3x.
    8. ABC at RT for 1 hour. Rinse in PBS 3x. ( ABC solution: 5ml PBS with 1 drop A and 1 drop B, mix well)
    9. DAB reaction: 2.5ml of distilled water + 1 drop Buffer + 2 drop DAB solution + 1 drop hydrogen peroxide. ~Needs 8 min to stop DAB reaction.
    10. Rinse in distilled water 3x.
    11. Counterstain and mount.

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Cell Culture Immunostaining of Osteocalcin:

    1. Wash with PBS 3x.
    2. Fixed in 4%PFA, 20 min at RT.
    3. Wash with PBS 3x.
    4. 0.5% Triton X-100 in PBS, 15 min at 4ºC.
    5. Wash with PBS 3x
    6. 1% BSA incubated for 30min at RT.
    7. 1:100, 1: 200, 1:300 dilution of Goat anti- osteocalcin, incubated at 4ºC, overnight.
    8. Wash with PBS 3x
    9. 1: 100 dilution of Rhodamine(Tritc) conjugated Donkey anti Goat IgG with 1%normal donkey serum+1%BSA in PBS, incubated at RT for 1 hr.
    10. Wash with PBS 3x
    11. Mount with 50% glycerol in PBS.

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ß-gal Staining:

Reagents:

    
 
0.1M Phosphate Buffer (pH 7.3):
    
  0.1M Sodium Phosphate monobasic
115ml
  
  0.1M sodium phosphate dibasic
385ml
  
 
Total Volume
500 ml
  
 
Fix solution:
 
 
0.5% Gluteraldehyde:
    
  25% gluteraldehyde
0.4 ml
  
 
100mM EGTA pH 7.3
2.5 ml
  
  1M magnesium chloride
0.1 ml
 
  0.1 M sodium phosphate pH 7.3
47.0 ml
  
 
Total Volume
50.0 ml
  
       
 
4% Paraformadehyde:
    
  paraformadehyde
20 g
 
  1M magnesium chloride
1ml
 
  100mM EGTA
25ml
 
  ~500 ml PBS    
  • Prepare fresh each time
 
Wash buffer:
    
  1M magnesium chloride
0.4 ml
  
  1% deoxycholate
2.0 ml
  
  2% Nonidet-P40
2.0 ml
  
  0.1M sodium phosphate pH 7.3
195.6 ml
  
 
Total Volume
200.0 ml
  
 
X-gal Staining:
    
  25mg/ml x-gal stock dissolved in di-methyl formamide
2.0 ml
  
  potassium ferrocyanide (Sigma P-9387)
0.106 g
  
  potassium ferricyanide (Sigma P-8131)
0.082 g
  
  wash buffer
48.0 ml
  
 
Total Volume
50 ml
  
  • This buffer can be reused, filter after use and store in the dark.
  • Also note, crystal from due to di-methyl formamide. If these crystal
    are a problem, prepare X-gal stock in dimethyl sulfoxide.
 


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X-gal Staining of Bone:

Whole mount staining:

  1. Dissect bone out of mice.
  2. Fix at tissue in fixative for 30min-2h at RT or overnight in 4ºC with 4% paraformadehyde.
  3. Rinse with PBS.
  4. Soak tissue in X-gal staining solution for 4 hours at 37ºC.
  5. Pour off the staining solution, replace with wash buffer, 2 times.
  6. Sock tissue in 30% sucrose in PBS.
  7. Embed and cut frozen section.


Frozen section staining:

    1. Bone after fixed and briefly rinse with PBS.
    2. Soak with decal solution. Gently rock at 4ºC for 1-3 days.
    3. Soak with 30% sucrose in PBS + 2mM MgCl2 at 4ºC for overnight.
    4. Embed in OCT.
    5. Cut frozen section. Place slides in wash solution.
    6. Incubate sections with x-gal solution at 37ºC for 1-3 hours (monitor staining every 30 min. It usually takes 2 hours).
    7. Rinse slides in PBS, 3 times.
    8. Rinse with 95% EtoH and stain with Eosin 20 secs for counterstain.
    9. Dehydrate and coverslip

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Fluorescent Label in Bone

Some fluorescent compounds, which are fixed in newly formed calcified tissue, are used to label bone deposition. Such labels help to determine the time sequence of bone growth. The label may be given single or double label by intraperitoneal injection.

Agents
Sigma Cat #
Color
Dosage (mg/kg)
Stock Con. (mg/ml)
Diluents
Calcein
C-0875
Green
10
3
2%NaHCO3
PH7.4
Xylenaol Orange(XO)
X-0127
Red
90
30
2%NaHCO3
PH7.4
Alizarin Complexone(AC)
A-3882
Red
30
10
2%NaHCO3
PH7.4
Oxytetracycline Hydrochloride(OH)
O-5875
Yellow
30
10
20% EtOH

 

Age
First Injection Before Sacrificing (days)
Second Injection Before Sacrificing (days)
<3 months
10
2
>3 months, <6 months
15
5
> 6 months
20
8

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June 21, 2004 2:02 PM    Page: