Nov 23, 2022
Staining of Gfap, Iba1,and NeuN on PFA-fixed mouse brain sections
- 1Université Laval
Protocol Citation: Daniel Manrique-Castano 2022. Staining of Gfap, Iba1,and NeuN on PFA-fixed mouse brain sections. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l27q5pg1y/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
Protocol established after several optimizing test
Created: November 23, 2022
Last Modified: November 23, 2022
Protocol Integer ID: 73191
Abstract
Staining protocol for Gfap, Iba1, and NeuN on PFA-fixed mouse brain sections.
Guidelines
Read the full protocol before starting the procedure.
Note that this protocol uses 3 hours (room temperature) incubation for primary antibodies.
Works with the listed antibodies and dilutions. For other references, preliminary tests are highly recommended.
Materials
Equipment
ImmEdge® Hydrophobic Barrier PAP Pen
NAME
Vector
BRAND
H-4000
SKU
LINK
Immunohistochemistry / Immunocytochemistry, Immunofluorescence, In situ hybridization
SPECIFICATIONS
Permeabilization solution = 0.3 % (v/v) TritonTriton X-100Sigma AldrichCatalog #T8787-50ML / = 0.1 % (v/v) TWEEN 20Sigma AldrichCatalog #P7949 /
0.3 Molarity (M) Glycine Contributed by users in 1x PBS
Antibody buffer = 0.1 % (v/v) TWEEN 20Sigma AldrichCatalog #P7949 / 1 % (v/v) Goat serumSigma AldrichCatalog #G9023 1 Mass / % volume Bovine Serum Albumin (BSA) Sigma AldrichCatalog #A7906
Primary antibodies:
GFAP Monoclonal Antibody (2.2B10)Thermo Fisher ScientificCatalog #13-0300
Anti Iba1 Rabbit antibodyFUJIFILM Wako Pure Chemical CorporationCatalog #019-19741
Anti-NeuN AntibodyEmd MilliporeCatalog #ABN91
Secondary antibodies:
Goat anti-Rat IgG (H L) Cross-Adsorbed Secondary Antibody Alexa Fluor 488Thermo Fisher ScientificCatalog #A-11006
Cy3-AffiniPure Goat Anti-Rabbit IgG (H L) antibodyJackson ImmunoresearchCatalog #111-165-003
Goat anti-Chicken IgY (H L) Cross-Adsorbed Secondary Antibody Alexa Fluor Plus 647Thermo Fisher ScientificCatalog #A32933 DAPI (46-Diamidino-2-Phenylindole Dilactate)Invitrogen - Thermo FisherCatalog #D3571
Mounting media:
Fluoromount-GElectron Microscopy SciencesCatalog #17984-25
Protocol materials
Triton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787-50ML
Materials
Goat anti-Chicken IgY (H L) Cross-Adsorbed Secondary Antibody Alexa Fluor Plus 647Thermo Fisher ScientificCatalog #A32933
Materials
Glycine
Materials
GFAP Monoclonal Antibody (2.2B10)Thermo Fisher ScientificCatalog #13-0300
Materials
Anti-NeuN AntibodyMerck Millipore (EMD Millipore)Catalog #ABN91
Materials
Cy3-AffiniPure Goat Anti-Rabbit IgG (H L) antibodyJackson ImmunoResearch Laboratories, Inc.Catalog #111-165-003
Materials
Fluoromount-GElectron Microscopy SciencesCatalog #17984-25
Materials, Step 9
Goat anti-Rat IgG (H L) Cross-Adsorbed Secondary Antibody Alexa Fluor 488Thermo Fisher ScientificCatalog #A-11006
Materials
DAPI (46-Diamidino-2-Phenylindole Dilactate)Invitrogen - Thermo FisherCatalog #D3571
Materials
Bovine Serum Albumin (BSA) Merck MilliporeSigma (Sigma-Aldrich)Catalog #A7906
Materials, Step 4
TWEEN 20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P7949
In Materials, Materials and 3 steps
Goat serumMerck MilliporeSigma (Sigma-Aldrich)Catalog #G9023
Materials, Step 4
Anti Iba1 Rabbit antibodyFUJIFILM Wako Pure Chemical CorporationCatalog #019-19741
Materials
Safety warnings
DAPI is highly toxic. Handle it with care.
Tissue preparation and blocking
Tissue preparation and blocking
20m
20m
Take out sections from -80 and place them in an incubator/plate for 00:20:00 at 37 °C . This step is performed to ensure tissue attachment to the crystal slides.
20m
2. Draw a hydrophobic barrier on each slide using ImmEdge®Hydrophobic Barrier PAP Pen and let dry for about 00:10:00 minutes. This will prevent buffers from escaping the tissue area.
Note
There are other hydrophobic pens on the market. However, this one is recommended for its quality, consistency, and durability.
10m
To initially rehydrate and permeabilize the tissue, place the slides in a slide jar containing the permeabilization solution with shaking for 00:30:00
Note
The proposed permeabilization solution contains glycine, suitable to reduce PFA-related autofluorescence, especially in the 488 channel.
.
30m
To prevent unspecific binding, incubate brain sections in permeabilization solution containing 5 % (v/v) Goat serumSigma AldrichCatalog #G9023 and 1 Mass / % volume Bovine Serum Albumin (BSA) Sigma AldrichCatalog #A7906 for 01:00:00 at Room temperature .
Note
Please note that blocking serum must be chosen according to secondary antibody species. For the procedure depicted in this protocol, all secondary antibodies are raised in goat. However, donkey secondary antibodies will be also suitable.
Additionally, consider as well avoiding BSA when primary antibodies come from goat or sheep. BSA can generate unspecific background.
1h
Antibody incubation
Antibody incubation
3h
3h
When blocking is finished, decant the buffer (no washing is required) and incubate primary antibodies in antibody buffer for 03:00:00 at Room temperature according to table 1.
| A | B | C | D | E | |
| Antibody | Company | Reference | Specie | Dilution | |
| Gfap | Invitrogen | 13-0300 | Rat | 1:500 | |
| Iba1 | Wako | 019-19741 | Rabbit | 1:500 | |
| NeuN | Millipore | ABN91 | Chicken | 1:300 |
Table 1. Primary antibodies
Note
Please note that the antibody buffers do not contain Triton X-100. The reason is that this detergent tends to break the hydrophobic barrier, and the sections may not be adequately incubated. In addition, because sufficient permeabilization has already been performed previously, this detergent is not contemplated in this step.
Please note that the proposed antibody incubation lasts only 3 hours. Although in most cases, staining protocols recommend overnight incubation for primary antibodies, the tests performed in our lab disclosed that, under the reported conditions, labeling for NeuN and Iba1 is superior at room temperature.
Also, the cited Iba1 antibody shows evident labeling and specificity superiority compared to others available in the market; therefore, highly recommended.
3h
When primary antibody incubation is finished, wash the sections 00:05:00 5x with 0.05 % (v/v) TWEEN 20Sigma AldrichCatalog #P7949 in PBS.
5m
Incubate secondary antibodies in 0.1 % (v/v) TWEEN 20Sigma AldrichCatalog #P7949 for 01:00:00 at Room temperature according to table 2.
| A | B | C | D | E | |
| Antibody | Company | Reference | Channel | Dilution | |
| Goat anti-rat | Invitrogen | A11006 | 488 | 1:500 | |
| Goat anti-rabbit | Jackson | 111-165-003 | Cy3 | 1:500 | |
| Goat anti-chicken | Invitrogen | A32933 | 647 | 1:500 | |
| Dapi | Invitrogen | D3571 | 405 (Dapi) | 1:5000 |
Table 2. Secondary antibodies
1h
When secondary antibody incubation is finished, wash the sections 00:05:00 3x with 0.05 % (v/v) TWEEN 20Sigma AldrichCatalog #P7949 in PBS. Follow this with 00:05:00 2x washes with PBS to remove all detergent traces.
10m
Clean the remaining buffer on the slides using absorbent tissue and mount the sections with a drop floFluoromount-GElectron Microscopy SciencesCatalog #17984-25 .