Methods and Techniques Discussion => Immunohistochemistry (IHC) => Topic started by: ihcwor2 on March 15, 2003, 01:54:15 AM

Title: Cytokeratin20 Immunoenzyme Protocol for Paraffin Sections
Post by: ihcwor2 on March 15, 2003, 01:54:15 AM
Cytokeratin20 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.   Antigen Retrieval Solution:
10mM Sodium Citrate Buffer:
To prepare 1000 ml,
Sodium citrate ------------- 2.94 g
Distilled water ------------- 1000 ml
Adjust pH to 6.0

C.   3% Hydrogen Peroxide:
To prepare 100 ml,
30% H2O2 ---------------- 10 ml
PBS or methanol ------- 90 ml

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

E.   Primary Antibody:
Mouse anti-cytokeratin20 (Dako, Cat# M-7019). Optimal dilution 1:80 in PBS.

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

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

H.   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.


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 steamer-citrate buffer antigen retrieval method.
6.   Rinse sections in PBS for 1x5min.

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

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

11.   Primary antibody: incubate sections with mouse anti-cytokeratin20 diluted 1:80 in PBS for 1 hour at room temperature.
12.   Rinse in PBS 3x5 min.

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

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

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

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

23.   Coverslip with mounting medium.


1.   Cytokeratin20 positive staining ---------------- brown
2.   Nuclei ------------------------------------------------- blue