Abcam Ad

Author Topic: BrdU Immunoenzyme Staining Protocol for Paraffin Sections  (Read 3806 times)

0 Members and 1 Guest are viewing this topic.

Offline ihcwor2

  • Administrator
  • GoldMember
  • *****
  • Posts: 335
    • http://www.ihcworld.com/
BrdU Immunoenzyme Staining Protocol for Paraffin Sections
« on: March 15, 2003, 01:37:42 AM »
BrdU Immunoenzyme 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.   3% Hydrogen Peroxide:
To prepare 100 ml,
30% H2O2 ---------------- 10 ml
PBS or methanol ------- 90 ml

E.   Blocking Solution:
2% Normal Horse Serum in PBS:
To prepare 100 ml
Normal horse serum ------ 2 ml
PBS --------------------------- 98 ml
Mix to dissolve.

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

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

H.   ABC Reagent:
HRP-streptavidin (Vector Laboratories, Cat# HA-5004). Optimal dilution 1:400.

I.   DAB Reagent:
0.02% DAB and 0.003% H2O2 in PBS
To prepare 100 ml
DAB ----------------------- 20 mg
PBS ----------------------- 100 ml
Stir to dissolve. Add 10 ul of 30% H2O2 and filter. Use the solution immediately.

       
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.   Hydrogen Peroxide: incubate sections in 3% H2O2 in PBS (or methanol) for 10-15 minutes to block endogenous peroxidase activity.
9.   Rinse in PBS for 1x2min.

10.   Blocking: incubate sections with 2% normal horse serum in PBS for 20 minutes to block non-specific binding of secondary immunoglobulin.
11.   Rinse in PBS for 1x2min.                                                                        

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

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

16.   ABC: incubate sections with HRP-streptavidin reagent diluted 1:400 in PBS for 30 minutes at room temperature.
17.   Rinse in PBS for 3x5min.

18.   DAB: incubate sections with DAB solution for 2-10 minutes.
19.   Rinse in distilled water 2x5min.

20.   Counterstain with hematoxylin if desire.
21.   Rinse in distilled water 2x5min.
22.   Dehydrate through 95% ethanol for 5min, 100% ethanol for 2x5min.
23.   Clear in xylene for 2x5min.

24.   Coverslip with mounting medium.


Results

1.   BrdU positive staining ------------------ brown
2.   Nuclei --------------------------------------- blue

BrdU Immunoenzyme Staining Protocol for Paraffin Sections
« on: March 15, 2003, 01:37:42 AM »