Detecting Abnormal Prion Proteins in Brain Tissue Using Immunohistochemistry

Overview

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

1. Cut sections of formalin-fixed, paraffin-embedded (FFPE) tissues at 3-5 µm thickness using a microtome.
2. 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.
3. Incubate the slides overnight at 50 ˚C to enhance the slide adhesion of tissues.
   NOTE: It is preferable to prepare freshly sectioned tissues for IHC protocols, although TSE positive and negative control sections can be prepared in advance and stored. Steps 2 to 4 must be performed in a chemical fume hood.

2. Deparaffinization and rehydration

1. Place the slides with the tissue sections into a stainless-steel staining basket (see Table of Materials). Immerse the basket into a xylene bath (stainless-steel staining container) for 3 min, remove and immerse once again.
2. Remove xylene and rehydrate sections by immersing them in an absolute ethanol bath for 3 min. Remove and immerse once again.
3. Air dry the sections and place in a 90% ethanol bath for 1 min. Transfer to a 70% ethanol bath for another minute, followed by a final transfer to a 50% ethanol bath for 1 min. For each ethanol bath treatment, gently agitate the slide basket twice and drain the basket prior to the next transfer.

3. Epitope retrieval

1. Carefully immerse the tissue sections in 98% formic acid at room temperature for 30 min. Rinse the sections in tap water for 5 min, followed by rinsing twice in distilled water.
   CAUTION: Formic acid is highly corrosive. Protective goggles and gloves must be worn.
2. Perform Heat-Induced Antigen Retrieval (HIER) following the steps below.
   NOTE: This step is performed by hydrated autoclaving at 121 °C for 30 min in a specific pressure chamber (see Table of Materials).
   1. Pre-heat the citrate buffer (10 mM, pH 6.1, see Table of Materials) at 98 °C for about 20 min in a stainless-steel staining container placed inside the pressure chamber filled with 500 mL of distilled water.
      NOTE: For the present study, the program Set Point was 1 (SP1) for the specific equipment used.
   2. After the alarm indicates that the equipment attained the programmed time and temperature, immerse the basket with slides and initiate the program to Set Point 2 (121° C for 30 min). For quality control purposes, place a section of adhesive autoclave indicator tape on the basket to monitor the temperature and pressure, and record the initial and final pressure of this program.
   3. If the number of slides to be immunostained does not reach the basket's capacity, use clean, blank slides to occupy any empty positions. After the program has terminated, allow the passive return of the chamber to ambient pressure (at least 30 min).
      NOTE: Longer durations may reduce non-specific background staining.
   4. Carefully transfer the slide basket to a stainless-steel staining container filled with distilled water for 5 min.

4. Inactivation of endogenous peroxidase

1. Immerse the slide basket containing the sample sections in a bath of 3% hydrogen peroxide (H₂O₂) in methanol for 30 min. Rinse the sections under running water (5 min). Drain and immerse the sections in 1x Tris-buffered saline (TBS) for an additional 5 min.

5. Immunodetection

NOTE: For the present study, immunodetection was executed using the capillary gap format using a commercially available slide clip assembly system (see Table of Materials). Other immunohistochemistry slide systems are also applicable.

1. Place each slide onto a commercially available cover-plate holder (see Table of Materials) pre-moistened with TBS, avoiding bubbles, with the tissue side facing the holder and slide edges coinciding with the two lower points of the holder.
   1. Hold the slide-holder assembly between the thumb and the forefinger, with one finger on top of the sample slide and the other on the bottom of the holder. Then, place the assembly in the gallery of the system.
   2. To ensure the set is well assembled, fill the well between the sample slide and the holder with TBS, which must not immediately overflow. From this point on, ensure that approximately 80 µL of TBS must be retained between the holder and the slide. Do not allow the sections to dry.
2. Once in the system, to decrease background staining prior to the treatment with primary antibody (antibody against prion proteins (anti-PrP), see Table of Materials), pre-incubate the sample slides with 20% normal serum from the same species as the secondary antibody host being used for immunostaining (in the present case, horse serum) for 30 min in TBS.
3. Thaw the number of aliquots of antibody to be used according to the animal species under analysis, the number of sample sections to be examined, and the amount of working dilution.
   NOTE: For best results, use 10% normal serum from the same species as the source of the secondary antibody and primary antibody solutions. If possible, for best results, the host source of the normal serum and secondary antibody should be from the same species.
4. Without washing the sections, apply the primary antibody solution directly (200 µL) in each well of the slide-holder set and incubate for 60 min at room temperature.
5. For washing, fill the wells of the slide-holder sets with TBS and wait for 5 min. Repeat twice.
6. Dilute the biotinylated secondary antibody (monoclonal anti-murine Horse Ab, see Table of Materials) at 1/200 in TBS with 10% horse serum. Prepare the volume required depending on the number of sections to be treated.
7. Apply the secondary antibody solution (200 µL) to each well of the slide-holder set. Incubate for 30 min at room temperature.
8. Wash as described in step 5.5.
9. For incubation with avidin-biotin complex peroxidase (ABC/HRP complex, see Table of Materials), prepare the reagent 30 min before use. Apply the ABC/HRP complex solution (200 µL) in each well of the slide-plate holder set. Incubate for 30 min at room temperature.
10. Wash as described in step 5.5.

6. Development with 3,3' diaminobenzidine (DAB) chromogen

CAUTION: DAB is a potential carcinogen. As a result, appropriate care is necessary while working with this reagent, including eye protection, lab coats, gloves, and good laboratory procedures. Dispose of following local regulations.

1. Dilute chromogen according to the manufacturer's instructions (see Table of Materials) immediately before use. Apply the chromogen solution (400 µL) to each well of the slide-plate set.
2. Incubate for up to 30 min at room temperature. In PrP^Sc (abnormal isoform of prion proteins) positive sections, a 10 min incubation period is usually sufficient.
3. Remove the residual chromogen solution by washing the slides in distilled water. Remove the slides from the coverplate holders and place them in a plastic container with distilled water.

Materials

Name	Company	Catalog Number	Comments
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