Immunohistochemistry Protocol for Paraffin-embedded Tissues

Solutions and reagents

Protocol

Troubleshooting


Solutions and reagents

1. Xylene

2. Ethanol, anhydrous denatured, histological grade (100%, 95%, 85%, 75%)

3.Washing buffer:

1XPBST 1 L
10X PBS 100 mL
dH2O 900 mL
Tween-20 1 mL
10X PBS 1 L
NaH2PO4•2H2O 2.84 g
Na2HPO4•12H2O 27.2 g
NaCl 90 g
dH2O 1000 mL

The pH should be about 7.2. Adjust if necessary with 1 M NaOH or 1 M HCl

4. Antigen Retrieval Solution:

A. 10mM Sodium Citrate Buffer, pH 6.0

C6H8O7•H2O 0.21g
C6H8Na3O7•2H2O 0.21g
dH2O 1000 mL

Adjust pH to 6.0

B. 10mM Tris Buffer with 1mM EDTA,pH 8.0 or 8.5 or 9.0

Tris 1.21 g
EDTA 0.37 g
dH2O 1000 mL

Adjust pH* to 8.0 or 8.5 or 9.0

*Note: Please refer to the antibody for individual antigen retrieval buffer and working conditions.

5. 3% Hydrogen Peroxide

6. Hematoxylin QS

7. Permanent Mounting Medium


Protocol

A. Deparaffinization and Rehydration


step1_1

  1. Heat slides in an oven at 60 °C for 5 min.
  2. Wash slides 3 times for 10 min each in xylene.
  3. Wash slides in 95% ethanol, 1 min.
  4. Wash slides in 85% ethanol, 1 min.
  5. Wash slides in 75% ethanol, 1 min.
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step1_2


  1. Rinse slides for 5 min in distilled water.

B. Antigen Retrieval*


step2

  1. Put the slides in Antigen Retrieval Solution and keep 120℃ for 2.5 min in pressure cooker.
  2. Then cool down at room temperature.
  3. *Note: Please refer to the antibody for individual antigen retrieval buffer and working conditions.

  4. Wash slides three times with distilled water (2 min each).

C. Staining


step3_1

  1. Inactivate endogenous peroxidase by covering tissue with 3% hydrogen peroxide for 15 min.
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    Wash slides twice with 1X PBST (2 min each).

step3_2

  1. Dilute primary antibody in the IHC Antibody Diluent per recommendation on the data sheet.
  2. Apply primary antibody to each section and incubate 90 min at room temperature*. Make sure the primary antibody solution covers the tissue evenly.
  3. *Note: Please refer to the antibody for individual buffer and working conditions.


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               Wash slides three times with 1X PBST (2 min each).

step3_3

  1. Apply to each section secondary antibody and incubate for 15 min at room temperature*.
  2. *Note: Please refer to the antibody for individual buffer and working conditions.


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               Wash slides three times with 1X PBST (2 min each).


step3_4

  1. Add freshly prepared DAB substrate to the sections.
  2. Incubate tissue sections with the substrate at room temperature until suitable staining develops (generally about 5 min).

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    Rinse sections with water.

step3_5


  1. Counterstain with Hematoxylin QS for 3 min
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    Rinse sections with water.


step3_6

  1. Wash slides in 75% ethanol, 1 min
  2. Wash slides in 85% ethanol, 1 min
  3. Wash slides in 95% ethanol, 1 min
  4. Wash slides in 2 changes of 100% ethanol rinses, 1 min each
  5. Wash slides in 3 changes of xylene, 1 min each
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step3_7


  1. Mount coverslips on slides using Permanent Mounting Medium
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step3_8


  1. Allow slides to dry overnight at room temperature and then analyze the results with microscope


Example

example_1

IHC staining of OriGene’s MKI67 UltraMAB (clone UMAB107) of normal human tonsil tissue. (UM500008).

example_2

IHC staining of OriGene’s MKI67 UltraMAB (clone UMAB107) of normal human tonsil tissue. (UM800033).




Useful links:

View our list of over 140,000 human tissues

View our list of the Specific Antibodies for Anatomic Pathology

View our list of detection kits developed by GBI Labs, Inc