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Author Topic: Factor VIII Immunofluorescence Method for Paraffin Sections  (Read 2418 times)

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Offline ihcwor2

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Factor VIII Immunofluorescence Method for Paraffin Sections
« on: March 15, 2003, 01:57:01 AM »
Factor VIII Immunofluorescence Staining Protocol for Paraffin Sections


Solutions and Reagents

A.   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

B.   Antigen Retrieval Solution:
      Trypsin Stock Solution (1%):
Trypsin --------------------------- 100 mg
             Distilled water ------------------ 10 ml
             Mix to dissolve. Aliquot and store at 20 C

             Calcium Chloride Stock Solution (1%):
Calcium chloride -------------- 100 mg
Distilled water ------------------ 10 ml

             Trypsin Working Solution (0.1%):
Trypsin stock solution (1%) ----------------- 1 ml
Calcium chloride stock solution 1%) ------ 1 ml
Distilled Water ---------------------------------- 10 ml
Adjust pH to 7.8 with 1N NaOH.

C.   Blocking Solution:
2% Normal Goat Serum in PBS:
To prepare 100 ml
Normal goat serum -------- 2 ml
PBS ---------------------------- 100 ml
Mix to dissolve.

D.   Primary Antibody:
Rabbit anti-Factor VIII (Zymed Labs Cat# 18-0018). Optimal dilution 1:100 in PBS.

E.   Secondary Antibody:
      Goat anti-rabbit IgG (H+L), biotinylated (Vector Laboratories, Cat# BA-1000). Optimal dilution 1:400.

F.   FITC-Streptavidin Reagent:
Vector Laboratories, Cat# SA-5001. Optimal dilution 1:50 in PBS.

G.   PI Stock Solution (1mg/ml or 1.5 mM):
To prepare, add 1 mg PI (Propidium Iodide) to 1 ml distilled water. Store stock solution at 4 C (or aliquot and store at 20 C), protected from light.  When handled properly, solutions are stable for at least six month.

H.   RNase A Stock Solution (1mg/ml):
To prepare, add 1 mg RNase A to 1 ml distilled water. Aliquot and store at 20 C freezer.

I.   PI Working Solution (1 ug/ml PI and 10 ug/ml RNase A in PBS):
To prepare, add 2 ul PI Stock Solution and 20 ul RNase A Stock Solution to 2 ml PBS.

       
Procedure

1.   Deparaffinize sections in xylene for 3x5min.
2.   Hydrate with 100% ethanol for 2x5min.
3.   Hydrate with 95% ethanol for 2x5min.
4.   Rinse in distilled water.

5.   Antigen Retrieval: Use trypsin digestion antigen retrieval method.
6.   Rinse sections in PBS for 1x5min.

7.   Blocking: incubate sections with 2% normal goat serum in PBS for 20 minutes
8.   Rinse in PBS for 1x2min.

9.   Primary antibody: incubate sections with rabbit anti-Factor VIII diluted 1:100 in PBS for 1 hour at room temperature.
10.   Rinse in PBS 3x5 min.

11.   Secondary antibody: incubate sections with biotinylated goat anti-rabbit IgG diluted 1:400 in PBS for 30 minutes at room temperature.
12.   Rinse in PBS for 3x5min.

13.   Incubate sections in FITC-streptavidin for 30 minutes, protecting the slide from light.
14.   Rinse 3x5min in PBS.

15.   Counterstain with PI Working Solution for 20 minutes at 37 C.
16.   Rinse 3x5min in PBS.

17.   Coverslip with aqueous mounting medium and seal with nail polish.

18.   Observation using a fluorescence microscope with appropriate filters. Store slides in the dark at 4 C.


Results

1.   Factor VIII positive staining (endothelial cells) ------- green
2.   Nuclei ----------------------------------------------------------- red

Factor VIII Immunofluorescence Method for Paraffin Sections
« on: March 15, 2003, 01:57:01 AM »