Methods and Techniques Discussion => Immunohistochemistry (IHC) => Topic started by: ihcwor2 on March 15, 2003, 01:39:04 AM

Title: BrdU Immunofluorescence Protocol for Paraffin Sections
Post by: ihcwor2 on March 15, 2003, 01:39:04 AM
BrdU 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.   2N HCl:
      To prepare 100 ml,
      10N HCl ------------------------------------ 20 ml
      Distilled water ----------------------------- 80 ml

C.   0.1M Borate Buffer, pH 8.5:
To prepare 100 ml,
Sodium borate (MW 381.4) ----------- 3.8 g
Distilled water ---------------------------- 100 ml
Mix to dissolve and adjust pH to 8.5

D.   Blocking Solution:
2% Normal Horse Serum in PBS:
To prepare 100 ml
Normal horse serum ------- 2 ml
PBS ---------------------------- 100 ml
Mix to dissolve.

E.   Primary Antibody:
Mouse anti-BrdU (Roche, Cat# 1-299-964). Optimal dilution 1:100 in PBS.

F.   Secondary Antibody:
      Horse anti-mouse IgG (H+L), biotinylated (Vector Laboratories, Cat# BA-2000). Optimal dilution 1:400.

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

H.   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.

I.   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.

J.   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.   Denature DNA by incubating the sections in 2N HCl for 60 minutes at 37 C.
6.   Neutralize the acid by immersing the sections in 0.1M borate buffer for 2x5 min.
7.   Rinse the sections 3x5 min in PBS.

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

10.   Primary antibody: incubate sections with mouse anti-BrdU diluted 1:100 in PBS for 1 hour at room temperature.
11.   Rinse in PBS 3x5 min.

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

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

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

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

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


Results

1.   BrdU positive staining ------------------- green
2.   Nuclei ---------------------------------------- red