Feb 05, 2024

Immunohistochemical staining, vibratome sections

DOI
https://dx.doi.org/10.17504/protocols.io.eq2lypnkqlx9/v1
Immunohistochemical staining, vibratome sections
  • 1KULeuven
Joris Van Asselberghs
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DOI: https://dx.doi.org/10.17504/protocols.io.eq2lypnkqlx9/v1
Protocol Citation: Joris Van Asselberghs, Veerle Baekelandt 2024. Immunohistochemical staining, vibratome sections. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lypnkqlx9/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 06, 2020
Last Modified: May 31, 2024
Protocol  Integer ID: 44295
Keywords: vibratome, sections, brain, neurological, IHC, staining, Immunohistochemical, ASAPCRN, general procedures for immunohistochemical staining, immunohistochemical staining, vibratome section, using vibratome section, vibratome sections this protocol
Abstract
This protocol outlines general procedures for Immunohistochemical staining, using vibratome sections.
Attachments
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Materials
Reagents Needed
  • 3% H2O2 in 1 x PBS
  • 1 x PBS 0.1% Triton X-100
  • 1 x PBS 0.1% Triton X-100 + 10% serum (accordingly to the secondary Abs)
  • Primary Antibodies (TH Ab152 1/5000)
  • Biotinylated Secondary Antibodies (SAR 1/300)
  • Streptavidine-HRP complex (1/1000)
  • DAB + H2O2 (1.4 µl H2O2 for 5 ml filtered DAB solution)
• DAB solution: 10 mg DAB (=1 tablet) for 25 ml 0.05 TRIS (TBS) pH 7.6; dissolve and filter through 0.22 µm filter, add H2O2 just before
  • PBS or PBS + 0.1% Na Azide
  • ½ PBS + ½ AD
  • Ethanol:
• 70% ethanol
• 90% ethanol
• 100% ethanol
  • Histoclear II
  • DPX mountant

Consumables Needed
  • Coverslips
  • Slides

Equipment Needed
  • Wobbler
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
Before start
This protocol uses floating sections in 24-well plate in 1 X PBS .
Quench Endogenous Peroxidase Activity
20m
Incubate samples in 1 x PBS 3% H2O2, for 00:10:00 at Room temperature on the wobbler (to quench endogenous peroxidase activity) using 500 µL / well.

10m
Rinse with 0.1 % (v/v) Triton X-100 in 1x PBS .

Rinse with 0.1 % (v/v) Triton X-100 in 1x PBS for 00:05:00 on wobbler.

5m
Repeat: rinse with 0.1 % (v/v) Triton X-100 in 1x PBS for 00:05:00 on wobbler.

5m
Primary Antibody Incubation
40m
Add 250 µL Primary Abs in 1 x PBS 0.1% Triton X-100 + 10% serum (accordingly to the secondary Abs) Overnight at Room temperature on wobbler.
Note
Dilution: 1st AB: TH Ab152 1/5000

40m
Rinse with 0.1 % (v/v) Triton X-100 in 1x PBS .
Rinse with 0.1 % (v/v) Triton X-100 in 1x PBS for 00:05:00 on wobbler.
5m
Repeat: rinse with 0.1 % (v/v) Triton X-100 in 1x PBS for 00:05:00 on wobbler.
5m
Secondary Antibody Incubation
20m
Add 250 µL biotinylated Secondary Abs in 1 x PBS 0.1% Triton X-100 for 00:30:00 at Room temperature on wobbler.
Note
Dilution: 2nd AB: SAR 1/300

30m
Rinse with 0.1 % (v/v) Triton X-100 in 1x PBS .
Rinse with 0.1 % (v/v) Triton X-100 in 1x PBS for 00:05:00 on wobbler.
5m
Repeat: rinse with 0.1 % (v/v) Triton X-100 in 1x PBS for 00:05:00 on wobbler.
5m
Streptavidine-HRP complex Incubation
30m
Add 250 µL Streptavidine-HRP complex 1/1000 (in 1 x PBS 0.1% Triton X-100) for 00:30:00 at Room temperature on wobbler.
Note
Dilution: STRP-HRP 1/1000

30m
Rinse with 0.1 % (v/v) Triton X-100 in 1x PBS .
Rinse with 0.1 % (v/v) Triton X-100 in 1x PBS for 00:05:00 on wobbler.
5m
Repeat: rinse with 0.1 % (v/v) Triton X-100 in 1x PBS for 00:05:00 on wobbler.
5m
DAB + H2O2

Safety information
Work on DAB Bench!

Prepare DAB solution: 10 mg DAB (=1 tablet) for 25 mL 0.05 TRIS (TBS) pH 7.6 ; dissolve and filter through 0.22 µm filter, add H2O2 just before use! Or Vector SG (TH).
(Add 1.4 µL H2O2 for 5 mL filtered DAB solution ).
Add 250 µL DAB + H2O2 (ON DAB BENCH!). Allow reaction to proceed for a few minutes at Room temperature without wobbling.
Rinse with 0.1 % (v/v) Triton X-100 in 1x PBS .

Replace TBS Triton X-100 with PBS or 0.1 % (v/v) Sodium Azide in PBS in case sections are not to be mounted immediately. Store at 4 °C .
½ PBS + ½ AD Rinse
30m
Briefly rinse sections in ½ PBS + ½ AD and mount on gelatin coated microscopy slides. Allow to dry for 00:30:00 in flow or couple of hours on bench.

30m
Dehydration
25m
Dehydrate samples in 70 % (v/v) ethanol for 00:05:00 .
5m
Dehydrate samples in 90 % (v/v) ethanol for 00:05:00 .
5m
Dehydrate samples in 100 % (v/v) ethanol for 00:05:00 .
5m
Repeat: dehydrate samples in 100 % (v/v) ethanol for 00:05:00 .
5m
Replace ethanol with Histoclear II for 00:05:00 .
5m
Mounting
30m
Mount coverslips on top of the slides with DPX and allow to dry Overnight in the flow.
30m
Press out bubbles next morning.