Detecting Abnormal Prion Proteins in Brain Tissue Using Immunohistochemistry

Published: April 30, 2024

Abstract

Source: Orge, L., et al. Detection of Abnormal Prion Protein by Immunohistochemistry. J. Vis. Exp. (2023).

This video demonstrates a technique to detect misfolded prion protein in brain sections using immunohistochemistry. Upon treating the brain section with formic acid to denature prions and minimize infection risk, as well as unmasking the prion aggregates using heat-induced epitope retrieval, the sections are immunolabeled for the misfolded prion protein aggregates and observed under a microscope.

Protocol

1. Tissue sectioning and slide preparation Cut sections of formalin-fixed, paraffin-embedded (FFPE) tissues at 3-5 µm thickness using a microtome. Float sections onto purified water with a temperature approximately 10 ˚C below the melting point of the paraffin used. Lift the sections from the water onto specially treated microscope slides (see Table of Materials). Allow the water to drain thoroughly from the slides. Incubate the slides overnight …

Divulgations

The authors have nothing to disclose.

Materials

Absolute ethanol Labchem LB0507-9010 Undiluted
               Diluted 90%, 70% and 50% in distilled water
Avidin-biotin complex and peroxidase
Vectastain Elite ABC kit Peroxidase
Vector Laboratories PK-6100 Prepare and gently mix 30 min before use according to kit instructions. Do not mix after standing.
Biotinylated secondary antibody (Horse anti-mouse IgG H+L) Vector Laboratories BA-2000-1.5 Dilute at 1/200 in TBS with 10% horse normal serum. Prepare the volume required depending on the number of sections.
Chromogen Diaminobenzidine- DAB, substrate kit, Peroxidase Vector Laboratories SK-4100 Prepare before use according to kit instructions.
Use 400 µL of solution per section.
DakoCytomation Pascal pressure chamber DAKO S2800
Ehrlich's Hematoxylin:
Absolute ethanol Labchem LB0507-9010
Glacial acetic acid Merck 101830
Potassium alum Merck 1.01047.1000
Glycerin Merck 1.04091.1000
Endogenous Peroxidase Block solution (3% concentration H2O2): 40 mL Hydrogen peroxide (30% w/w) in 360 mL Methanol.
Prepare before use
Hydrogen peroxide (30% w/w) Scharlau HI0136
Methanol Sigma Aldrich 322415-2L
Formic acid 98% Merck 1.00264.1000 Undiluted
Microtome Shandon-AS325 Microtome Shandon-AS325
Normal serum (20% ) block solution in TBS:
Horse normal serum
Gibco 16050-122 Prepare final volume according to the number of sections in the assay
(200 µL of solution per section).
Primary antibody anti-PrP Mouse MAb 2G11 BIORAD MCA2460 PrP 146-R154R171182
Ovine including atypical scrapie, cervine, feline. Not suitable for bovine.
According to the number of sections in the assay (200 µL of solution per section) and antibody dilution, prepare final volume in TBS supplemented with 10% of normal serum from the species the secondary antibody was raised in (horse normal serum)
Usual antibody dilution: MAb 2G11 1/100 but working dilution should be established in every new batch to get the concentration to give the strongest labelling with lowest background. For storage, freeze aliquot volumes of a minimum of 10 μL into sterile microtubes. Defrost and use one aliquot at a time.
Primary antibody anti-PrP Mouse MAb 12F10 Cayman Chemical Company 189710 PrP142-160
Bovine, not suitable for ovine
Usual antibody dilution: 1/200 but working dilution should also be established. Prepare as MAb 2G11
Shandon CoverplateTM chamber Thermo Scientific 72110017
Shandon Sequenza® Immunstaining center Thermo Scientific 73300001
Shandon Sequenza® Immunstaining slide rack Thermo Scientific 73310017
Solution Citrate Buffer (10 mM pH 6.1): 2.55 g Tri-sodium citrate dihydrate and 0.255 g Citric acid in one litre purified water.
Adjust pH of working solution to 6.1 using 10 mM citric acid solution (1.05 g citric acid in 500 mL purified water)
Prepare on assay day.
Tri-sodium citrate dihydrate Sigma-aldrich S4641-500G
Citric acid Sigma Aldrich C0759
Staining jar and basket Deltalab 19360
19361
Superfrost Plus microscope slides VWR 631-0108
Tris-Buffered Saline solution (TBS) (50 mM TRIZMA BASE; 0.8% NaCI; pH 7.6): 10xTBS (stock solution 0.5 M TRIZMA BASE; 8% NaCI; pH 7.6):
TRIZMA BASE 60,57 g and NaCl 80 g in 800 mL purified water. Adjust pH of stock solution using Hydrochloric acid 37% and final volume to one litre with purified water (keep 5± 3 °C until 2 months)
Dilute TBS stock solution 1/10 on assay day.
TRIZMA BASE Sigma Aldrich T6066-1KG
Sodium Chloride (NaCl) Merck 106404
Xylene Panreac Applied Chem ITW reagents 251769 Undiluted

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Citer Cet Article
Detecting Abnormal Prion Proteins in Brain Tissue Using Immunohistochemistry. J. Vis. Exp. (Pending Publication), e22123, doi: (2024).

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