Immunohistochemical coloring of Paraffin Embedded Tissue Samples

The procedure is developed for use at the Department of Clinical Research, Hvidovre Hospital.
(The procedure will be performed at the pathological department, Hvidovre Hospital).
The aim of this instruction is to insure the correct immunohistochemical coloring of paraffin embedded tissue samples.

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Materials:

• TBS buffer (Purchased by the Pathological Department)
• TBST buffer (Purchased by the Pathological Department)
• Tris-EDTA buffer (TE-buffer, pH 9,00) (Purchased by the Pathological Department)
• 0,5% CuSO₄ (in TBS buffer)
• Dako's Envision Linked kit (containing secondary antibody - rabbit Anti-Mouse, HP-Blocking reagent, Envision reagent, DAB and substrate buffer)
• Hematoxylin (Purchased by the Pathological Department)
• Primary antibodies: For the MMR staining use:
  • MLH1: BD BioScience, Cat. No. 550838. Conc. 1:100
  • PMS2: BD BioScience, Cat. No. 556415. Conc. 1:500
  • MSH2: NovoCastra, Cat. No. 138611. Conc. 1:100
  • MSH6: BD BioScience, Cat. No. 610919. Conc. 1:200
• Microwave (use the one at the Pathological Department, program 3)
• 99,5% Ethanol (Purchased by the Pathological Department)
• 96% Ethanol (Purchased by the Pathological Department)
• 70% Ethanol (Purchased by the Pathological Department)
• Albumin-Bovin opløsning (Purchased by the Pathological Department)
• Coloring trays
• Pipettes

Procedure:

(The minimal volume pr. slide is 200ml)

• Dry the slides over night at 37 degrees
• Wash in tissue-clear for 2 x 5 minutes
• Rinse in 99,5% ethanol for 5 minutes
• Rinse in 95% ethanol for 5 minutes
• Rinse under the faucet for 5 minutes
• Place the slides in 250 ml TE-buffer, pH 9.00
• Boil in microwave (program 3)
• Leave the slides in the hot buffer for 20 minutes
• Rinse under the faucet for 5 minutes
• Rinse in TBST for 5 minutes
• Circle the tissue with a "Dakopen"
• Incubate in primary antibody for 25 minutes
• Rinse in TBST for 5 minutes
• Incubate in secondary antibody for 20 minutes
• Rinse in TBST for 5 minutes
• Incubate in HP Blocking reagent for 7,5 min
• Rinse in TBST for 5 minutes
• Incubate in Envision reagent for 25 minutes
• Rinse in TBST for 5 minutes
• Incubate in DAB for 15 minutes
• Rinse under the faucet for 5 minutes
• Incubate in Hematoxyline for 2 minutes
• Rinse under the faucet for 10 minutes
• Intensify by incubating in 0,5 % CuSO₄ solution for 5 minutes
• Rinse under the faucet for 5 minutes
• Dehydrate (moving slides from 96% Ethanol to clean 99,5% Ethanol)
• Cover slides with glass using the "Tissue-Tek machine" or manually with pertex

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