Staining of CD31 and CD13 in PDGFR-B/Td-tomato brain sections
Daniel Manrique-Castano
Abstract
This protocol is suitable for the staining of CD31 and CD13 in PDGFR-B/Td-Tomato fixed mouse brain sections.
Before start
Steps
Tissue preparation and blocking
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 1h 0m 0s at Room temperature .
Antibody incubation
When blocking is finished, aspirate the buffer (no washing is required) and incubate CD31 (BD Bioscience, 550274, 1:200) in antibody buffer for 3h 0m 0s at 4°C.
When primary antibody incubation is finished, aspirate the media and wash the sections 0h 5m 0s with 0.05% (v/v)
Incubate Donkey α Goat 488 (Invitrogen, A-11055, 1:500) secondary antibody in 0.1% (v/v) 1h 0m 0s at Room temperature
When secondary antibody incubation is finished, aspirate the media and wash the sections 0h 5m 0s with 0.05% (v/v)
Tissue blocking
Perform a second blocking step using 5% Donkey serum in 0.1% (v/v)
When blocking is finished, aspirate the buffer (no washing is required) and incubate CD13 (R&D, AF2335, 1:300) in antibody buffer for 3h 0m 0s at 4Room temperature.
When primary antibody incubation is finished, aspirate the media and wash the sections 0h 5m 0s with 0.05% (v/v)
Incubate Donkey α Rat 647 (Jackson, 712-605-153, 1:300) secondary antibody in 0.1% (v/v) 1h 0m 0s at Room temperature. Add DAPI (Invitrogen, D3571, 1:5000)
Wash and mounting
When secondary antibody incubation is finished, wash the sections 0h 5m 0s with 0.05% (v/v) 0h 5m 0s washes with PBS to remove all detergent traces.
Clean the remaining buffer on the slides using absorbent tissue and mount the sections with a drop of
