Sep 05, 2023
Staining of CD31 and CD13 in PDGFR-B/Td-tomato brain sections
- 1Université Laval
Protocol Citation: Daniel Manrique-Castano 2023. Staining of CD31 and CD13 in PDGFR-B/Td-tomato brain sections. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9p2b1g3e/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 05, 2023
Last Modified: September 05, 2023
Protocol Integer ID: 87394
Keywords: Staining, GFAP, IBA1, Immunofluorescence
Abstract
This protocol is suitable for the staining of CD31 and CD13 in PDGFR-B/Td-Tomato fixed mouse brain sections.
Guidelines
Read the entire protocol before starting the procedure.
Note that this protocol uses free-floating sections to enhance CD31 staining.
This protocol uses sequential staining to avoid cross-reactivity between CD31 and CD13.
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
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) Normal Donkey SerumMerck MilliporeSigma (Sigma-Aldrich)Catalog #D9663-10ml 1 Mass / % volume Bovine Serum Albumin (BSA) Sigma AldrichCatalog #A7906
Primary antibodies:
Mouse Aminopeptidase N/CD13 AntibodyR&D SystemsCatalog #AF2335
Purified Rat Anti-Mouse CD31BD BiosciencesCatalog #550274
Secondary antibodies:
Donkey anti-Goat IgG (H+L) Thermo Fisher ScientificCatalog #A-11055
Alexa Fluor® 647 AffiniPure Donkey Anti-Rat IgG (H+L)Jackson ImmunoResearch Laboratories, Inc.Catalog #712-605-153
DAPI (46-Diamidino-2-Phenylindole Dilactate)Invitrogen - Thermo FisherCatalog #D3571
Mounting media:
Fluoromount-GElectron Microscopy SciencesCatalog #17984-25
Protocol materials
TWEEN 20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P7949
In Materials, Materials and 7 steps
Mouse Aminopeptidase N/CD13 AntibodyR&D SystemsCatalog #AF2335
Materials
Normal Donkey SerumMerck MilliporeSigma (Sigma-Aldrich)Catalog #D9663-10ml
Materials
Bovine Serum Albumin (BSA) Merck MilliporeSigma (Sigma-Aldrich)Catalog #A7906
Materials
Donkey anti-Goat IgG (H L) Thermo Fisher ScientificCatalog #A-11055
Materials
Purified Rat Anti-Mouse CD31BD BiosciencesCatalog #550274
Materials
DAPI (46-Diamidino-2-Phenylindole Dilactate)Invitrogen - Thermo FisherCatalog #D3571
Materials
Fluoromount-GElectron Microscopy SciencesCatalog #17984-25
Materials, Step 12
Glycine
Materials
Alexa Fluor® 647 AffiniPure Donkey Anti-Rat IgG (H L)Jackson ImmunoResearch Laboratories, Inc.Catalog #712-605-153
Materials
Triton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787-50ML
Materials
Safety warnings
DAPI is highly toxic. Handle it with care.
Tissue preparation and blocking
Tissue preparation and blocking
20m
20m
Extract the sections from the anti-freeze media and rinse them in PBS using a 24-well plate.
Aspirate the PBS and incubate the sections in the Blocking solution for 01:00:00 at Room temperature .
1h
Antibody incubation
Antibody incubation
12h
12h
When blocking is finished, aspirate the buffer (no washing is required) and incubate CD31 (BD Bioscience, 550274, 1:200) in antibody buffer for Overnight at 4 °C .
Note
Since the sections are already permeabilized, please note that antibody buffers do not contain Triton X-100
Please note that the proposed antibody incubation includes only CD31. CD13 will be incubated after.
3h
When primary antibody incubation is finished, aspirate the media and wash the sections 00:05:00 5x with 0.05 % (v/v) TWEEN 20Sigma AldrichCatalog #P7949 in PBS.
5m
Incubate Donkey α Goat 488 (Invitrogen, A-11055, 1:500) secondary antibody in 0.1 % (v/v) TWEEN 20Sigma AldrichCatalog #P7949 for 01:00:00 at Room temperature
1h
When secondary antibody incubation is finished, aspirate the media and wash the sections 00:05:00 5x with 0.05 % (v/v) TWEEN 20Sigma AldrichCatalog #P7949 in PBS.
5m
Tissue blocking
Tissue blocking
Perform a second blocking step using 5% Donkey serum in 0.1 % (v/v) TWEEN 20Sigma AldrichCatalog #P7949 in PBS.
Antibody incubation
Antibody incubation
12h
12h
When blocking is finished, aspirate the buffer (no washing is required) and incubate CD13 (R&D, AF2335, 1:300) in antibody buffer for 03:00:00 at Room temperature .
Note
Please note that this antibody incubation is performed differently from the first one (3 hours at room temperature).
3h
When primary antibody incubation is finished, aspirate the media and wash the sections 00:05:00 5x with 0.05 % (v/v) TWEEN 20Sigma AldrichCatalog #P7949 in PBS.
5m
Incubate Donkey α Rat 647 (Jackson, 712-605-153, 1:300) secondary antibody in 0.1 % (v/v) TWEEN 20Sigma AldrichCatalog #P7949 for 01:00:00 at Room temperature . Add DAPI (Invitrogen, D3571, 1:5000)
1h
Wash and mounting
Wash and mounting
12h
12h
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 of Fluoromount-GElectron Microscopy SciencesCatalog #17984-25 .