Sep 22, 2023
Immunohistochemistry/Immunofluorescence
- Michael Henderson1
- 1Van Andel Institute
Protocol Citation: Michael Henderson 2023. Immunohistochemistry/Immunofluorescence. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl89m9dv2w/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: January 04, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 56552
Keywords: immunohistochemisty, immunofluorescence, formic acid retrieval, Sudan Black Treatment, ASAPCRN
Abstract
This protocol details about the immunohistochemisty/immunofluorescence staining techniques for tissue.
Attachments
338-741.pdf
527KB
Materials
Solutions and Reagents
0.5 M Tris (8 L)
| A | B | C | |
| Needed (mL) | Stock Solution | Final Concentration | |
| 5 L | dH2O | ||
| 485 g | Tris base | 0.5 M | |
| 240 mL | Concentrated HCl | ||
| pH to 7.6 | |||
| To 8L | dH2O |
Reagents
| A | B | C | D | E | |
| Vendor | Catalog # | Qty | Unit Price | Description | |
| Vector Laboratories | H-3300 | 1 | 132.60 | Antigen Unmasking Solution, Citric Acid Based | |
| Vector Laboratories | H-4000 | 1 | 120.00 | ImmEdge Hydrophobic Barrier Pen | |
| Vector Laboratories | PK-6100 | 1 | 248.63 | VECTASTAIN Elite ABC Kit (Standard) | |
| Vector Laboratories | SK-4105 | 1 | 138.13 | ImmPACT DAB Peroxidase (HRP) Substrate | |
| Vector Laboratories | BA-2000 | 1 | 55 | Biotinylated Horse Anti-Mouse IgG Antibody | |
| Vector Laboratories | BA-1100 | 1 | 140 | Biotinylated Horse Anti-Rabbit IgG Antibody | |
| Sigma | 199664-25G | 1 | 66.60 | Sudan Black B | |
| Thermo Fisher | 6765001 | 1 | 46.41 | Shandon Harris Hematoxylin (non acidic) | |
| Fisher Scientific | 23-244-256 | 1 | 22.96 | Cytoseal 60; 4 oz. | |
| Southern Biotech | 0100-01 | 1 | 45.14 | DAPI Fluoromount-G |
Antigen Unmasking Solution Citrate-BasedVector LaboratoriesCatalog #H-3300
ImmEdge hydrophobic barrier pap penVector LaboratoriesCatalog #H-4000
VECTASTAIN Elite ABC HRP Kit (Peroxidase, Standard)Vector LaboratoriesCatalog #PK-6100
ImmPACT® DAB Substrate Peroxidase (HRP) Vector LaboratoriesCatalog #SK-4105
Horse Anti-Mouse IgG Antibody (H L) BiotinylatedVector LaboratoriesCatalog #BA-2000
Horse Anti-Rabbit IgG Antibody (H L) BiotinylatedVector LaboratoriesCatalog #BA-1100
Sudan black BSigma AldrichCatalog #199664
Shandon™ Harris Hematoxylin, NonacidifiedThermo FisherCatalog #6765001
Fluoromount-GSouthern BiotechCatalog #0100-01
Day 1
Day 1
2h 11m
2h 11m
Label slides with antibody and treatment to be used.
De-paraffinize slides in fresh xylenes, then in a descending ethanol series.
De-paraffinize slides 00:05:00 in fresh xylenes. (1/2)
5m
De-paraffinize slides 00:05:00 in fresh xylenes. (2/2)
5m
De-paraffinize slides in ethanol 100% for 00:01:00 .
1m
De-paraffinize slides in ethanol 100% for 00:01:00 .
1m
De-paraffinize slides in ethanol 95% for 00:01:00 .
1m
De-paraffinize slides in ethanol 80% for 00:01:00 .
1m
De-paraffinize slides in ethanol 70% for 00:01:00 .
1m
Formic Acid Retrieval (If necessary, do here).
Immerse slides in ddH2O for 00:01:00 , place in recycled FA for 00:05:00 and wash in running tap H2O for 00:10:00 ..
16m
Microwave antigen retrieval (CA; If necessary do here).
Dilute antigen unmasking solution (Vector Labs, citric acid) 1:100 in dH2O (2.5 mL /250 mL dH2O/boat).
Place in Biogenex EZ-Retriever microwave for 00:15:00 at 95 °C .
15m
Cool for 00:20:00 atRoom temperature .
20m
Wash slides for 00:10:00 in running tap H2O.
10m
Immerse in freshly prepared Methanol/H2O2 (150 mL Methanol + 30 mL stock 30% H2O2) 00:30:00 .
Note
DO NOT GET ON SKIN OR LEAVE SPILL ON BENCH.
Then, wash in running tap H2O for 00:10:00 .
Note
*This step is not necessary for immunofluorescence.
*May use DI water/H2O2 (150 mL DI water + 50 mL stock 30% H2O2).
40m
Wash in 0.1 Molarity (M) Tris buffer, 7.6 00:05:00 . Discard all Tris washes.
5m
Block in 0.1 Molarity (M) Tris/2% FBS (Tris/FBS) 00:05:00 +. Keep blocking solution for up to 2 weeks @ 4 °C .
5m
Dilute primary antibodies in Tris/FBS, and prepare humidified chamber(s) by soaking towel in the middle of the slide chamber(s).
Wipe excess fluid off back of slides and from around tissue and apply 200 µL of primary antibody to slides. Make sure antibodies cover all sections.
Note
Hydrophobic pen may be used at this point if desired, but CANNOT be used for immunofluorescence.
Incubate at 4 °C in humidified chamber Overnight .
5m
Day 2
Day 2
2h 11m
2h 11m
Rinse off antibody from tissue using Tris.
Note
Carefully direct spray from wash bottle around tissue, NOT directly on it.
Wash in Tris 00:05:00 .
5m
Block in Tris/FBS 00:05:00 .
5m
Dilute Vector biotinylated IgG 1:1000 in Tris/FBS and apply200 µL to wiped slides.
*For immunofluorescence, dilute fluorescent secondary antibodies 1:500 in Tris/FBS and apply 200 µL to wiped slides. Keep slides in the dark from here on. Incubate at 4 °C in humidified chamber overnight or at Room temperature for 03:00:00 or overnight at 4 °C . Proceed to Day 3.
3h
Incubate at Room temperature in humidified chamber 01:00:00 .
1h
Rinse biotinylated IgG using Tris.
Wash in Tris 00:05:00 .
5m
Block in Tris/FBS 00:05:00 .
5m
Mix AB solution (Vector peroxidase standard) in Tris/FBS to a dilution of 1:000 (ie add 1 µL of A and 1 µL of B to 1 mL of 0.1 Molarity (M) Tris/2%FBS). Vortex and let sit 00:15:00 before use. Then, apply 200 µL of AB solution to wiped slides.
15m
Incubate at Room temperature in humidified chamber for 01:00:00 .
1h
Rinse off AB using Tris.
Immerse in Tris 00:05:00 .
5m
Make Vector DAB solution (1 drop of DAB per mL of Stable DAB Buffer) .
Apply 200 µL of DAB to each slide and incubate until a visible brown signal is seen and well developed.
Note
Development time may differ by antibody, but all sections treated with the same antibody should be developed for the same amount of time.
Rinse with Tris and place in dH2O. Wash 00:05:00 in dH2O. Filter Harris hematoxylin.
5m
Counterstain briefly with Harris hematoxylin (~00:00:15 , depending on age).
15s
Wash in running tap H2O 00:05:00 .
5m
Dehydrate and clear in ascending ethanol and xylenes.
Dehydrate and clear in 70% ethanol for 00:01:00 and 70% xylenes for 00:05:00 .
6m
Dehydrate and clear in 80% ethanol for 00:01:00 and 80% xylenes for 00:05:00 .
6m
Dehydrate and clear in 95% ethanol for 00:01:00 and 95% xylenes for 00:05:00 .
6m
Dehydrate and clear in 100% ethanol for 00:01:00 and 100% xylenes for 00:05:00 .
6m
Dehydrate and clear in 100% ethanol for 00:01:00 and 100% xylenes for 00:05:00 .
6m
Coverslip with cytoseal.
Dry in tissue processor closet Overnight .
5m
Day 3 (Immunofluorescence)
Day 3 (Immunofluorescence)
16m 10s
16m 10s
Filter Sudan Black. This process can take a long time, so start early.
Rinse off AB using Tris.
Wash in running tap H2O for 00:05:00 .
5m
Wash in Tris for 00:05:00 in green boats.
5m
Sudan Black Treatment (0.3% Sudan Black B in 70% Ethanol)
Use a control slide (usually 1 positive primary and 1 secondary only) to titrate for background reduction without changing signal intensity (usually 00:00:10 to 00:01:00 ).
1m 10s
Image before and after Sudan black treatment for various times.
Treat all slides identically.
Wash in 0.1 Molarity (M) Tris 00:05:00 in green boats.
5m
Coverslip using non-photobleaching reagent (FluorMount with DAPI). Allow to dry completely before imaging on scanner.