Sep 01, 2023

Public workspaceStaining of GFAP and IBA1 in PDGFR-B/Td-tomato brain sections

DOI
dx.doi.org/10.17504/protocols.io.14egn36mml5d/v1
  • 1Université Laval
Daniel Manrique-Castano
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DOI: dx.doi.org/10.17504/protocols.io.14egn36mml5d/v1
Protocol Citation: Daniel Manrique-Castano 2023. Staining of GFAP and IBA1 in PDGFR-B/Td-tomato brain sections. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn36mml5d/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: September 01, 2023
Last Modified: September 01, 2023
Protocol Integer ID: 87251
Keywords: Staining, GFAP, IBA1, Immunofluorescence
Abstract
This protocol is suitable for the staining of GFAP and IBA1 in PDGFR-B/Td-Tomato fixed mouse brain sections.
Guidelines
Read the entire protocol before starting the procedure.

Note that this protocol uses 3 hours (room temperature) incubation for primary antibodies.

Do not submit the tissue to antigen retrieval or other acidic solutions to preserve theTd-tomato signal.

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
https://vectorlabs.com/products/histology/immedge-hydrophobic-barrier-pen
LINK
Immunohistochemistry / Immunocytochemistry, Immunofluorescence, In situ hybridization
SPECIFICATIONS
Permeabilization solution = Concentration0.3 % (v/v) TritonReagentTriton X-100Sigma AldrichCatalog #T8787-50ML / = Concentration0.1 % (v/v) ReagentTWEEN 20Sigma AldrichCatalog #P7949 /
Concentration0.3 Molarity (M) ReagentGlycine Contributed by users in 1x PBS

Antibody buffer = Concentration0.1 % (v/v) ReagentTWEEN 20Sigma AldrichCatalog #P7949 / Concentration1 % (v/v) ReagentNormal Donkey SerumMerck MilliporeSigma (Sigma-Aldrich)Catalog #D9663-10ml Concentration1 Mass / % volume ReagentBovine Serum Albumin (BSA) Sigma AldrichCatalog #A7906

Primary antibodies:

ReagentGFAP Monoclonal Antibody (2.2B10)Thermo Fisher ScientificCatalog #13-0300
ReagentAnti Iba1 Rabbit antibodyFUJIFILM Wako Pure Chemical CorporationCatalog #019-19741


Secondary antibodies:
ReagentAlexa Fluor® 647 AffiniPure Donkey Anti-Rat IgG (H L)Jackson ImmunoResearch Laboratories, Inc.Catalog #712-605-153
ReagentAnti-RabbitInvitrogen - Thermo FisherCatalog #A21206
ReagentDAPI (46-Diamidino-2-Phenylindole Dilactate)Invitrogen - Thermo FisherCatalog #D3571

Mounting media:

ReagentFluoromount-GElectron Microscopy SciencesCatalog #17984-25

Protocol materials
ReagentNormal Donkey SerumMerck MilliporeSigma (Sigma-Aldrich)Catalog #D9663-10ml
Materials, Step 4
ReagentFluoromount-GElectron Microscopy SciencesCatalog #17984-25
Materials, Step 9
ReagentTriton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787-50ML
Materials
ReagentGFAP Monoclonal Antibody (2.2B10)Thermo Fisher ScientificCatalog #13-0300
Materials
ReagentAnti-RabbitInvitrogen - Thermo FisherCatalog #A21206
Materials
ReagentDAPI (46-Diamidino-2-Phenylindole Dilactate)Invitrogen - Thermo FisherCatalog #D3571
Materials
ReagentTWEEN 20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P7949
In Materials, Materials and 3 steps
ReagentBovine Serum Albumin (BSA) Merck MilliporeSigma (Sigma-Aldrich)Catalog #A7906
Materials, Step 4
ReagentAlexa Fluor® 647 AffiniPure Donkey Anti-Rat IgG (H L)Jackson ImmunoResearch Laboratories, Inc.Catalog #712-605-153
Materials
ReagentAnti Iba1 Rabbit antibodyFUJIFILM Wako Pure Chemical CorporationCatalog #019-19741
Materials
ReagentGlycine
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 Duration00:20:00 at Temperature37 °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 Duration00:10:00 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 Duration00:30:00
Note
The proposed permeabilization solution contains glycine, which is suitable for reducing PFA-related autofluorescence, especially in the 488 channel.

Antigen retrieval or any treatment with acid is not recommended given that it destroys the endogenous Td-tomato signal from PDGFR-B+ cells.
.

30m
To prevent unspecific binding, incubate brain sections in permeabilization solution containing Concentration5 % (v/v) ReagentNormal Donkey SerumMerck MilliporeSigma (Sigma-Aldrich)Catalog #D9663-10ml and Concentration1 Mass / % volume ReagentBovine Serum Albumin (BSA) Sigma AldrichCatalog #A7906 for Duration01:00:00 at TemperatureRoom 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 donkey. However, goat secondary antibodies will also be suitable.

Additionally, consider avoiding BSA when primary antibodies come from goat or sheep. BSA can generate unspecific background.



1h
Incubation
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 Duration03:00:00 at TemperatureRoom temperature according to table 1.


ABCDE
AntibodyCompanyReferenceSpecieDilution
GfapInvitrogen13-0300Rat1:500
Iba1Wako019-19741Rabbit1:500
Table 1. Primary antibodies

Note
Please note that antibody buffers do not contain Triton X-100. This detergent tends to break the hydrophobic barrier and the sections may not be adequately incubated. In addition, given that the sections are already permeabilized, we do not contemplate the use of this detergent.

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 showed better efficiency at room temperature.

The indicated Iba1 antibody is superior to others available in the market.

3h
Incubation
Pipetting
When primary antibody incubation is finished, wash the sections Duration00:05:00 5x with Concentration0.05 % (v/v) ReagentTWEEN 20Sigma AldrichCatalog #P7949 in PBS.
5m
Wash
Incubate secondary antibodies in Concentration0.1 % (v/v) ReagentTWEEN 20Sigma AldrichCatalog #P7949 for Duration01:00:00 at TemperatureRoom temperature according to table 2.
ABCDE
AntibodyCompanyReferenceChannelDilution
Donkey anti-ratJackson712-605-1536471:500
Donkey anti-rabbitInvitrogenA212064881:500
DapiInvitrogenD3571405 (Dapi)1:5000
Table 2. Secondary antibodies

1h
Incubation
Pipetting
When secondary antibody incubation is finished, wash the sections Duration00:05:00 3x with Concentration0.05 % (v/v) ReagentTWEEN 20Sigma AldrichCatalog #P7949 in PBS. Follow this with Duration00:05:00 2x washes with PBS to remove all detergent traces.

10m
Wash
Clean the remaining buffer on the slides using absorbent tissue and mount the sections with a drop floReagentFluoromount-GElectron Microscopy SciencesCatalog #17984-25 .