Nov 24, 2021
Emir's methanol fixation IF protocol
This protocol is a draft, published without a DOI.
- 1MDC Berlin
- AG Gerhardt
Protocol Citation: Emir Bora Akmeriç 2021. Emir's methanol fixation IF protocol. protocols.io https://protocols.io/view/emir-39-s-methanol-fixation-if-protocol-b2bzqap6
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: November 24, 2021
Last Modified: November 24, 2021
Protocol Integer ID: 55385
Abstract
Methanol fixation protocol for immunofluorescence, should hopefully work for Claudin-5 staining.
Protocol materials
MethanolP212121Catalog #PA-33900HPLCCS4L
Safety warnings
Methanol is a toxic substance, work under the hood at all times!
Fixation
Fixation
45m
45m
Bring methanol to -20C and prepare compartment in cell culture freezer to quickly transfer slides/dishes. Get a box of ice-20 °C MethanolP212121Catalog #PA-33900HPLCCS4L
5m
Incubate sample in methanol for 12 minutes.
12m
Wash samples with PBS 3 times on ice
5m
Incubate samples with PBS on ice for 5 minutes, repeat 3 times
18m
Store samples in the fridge until further use4 °C
Blocking/Staining
Blocking/Staining
19h 33m
19h 33m
Block for 1hr in 5% normal goat serum diluted in PBS
1h
Make primary antibody mix for the next step in the meantime
Incubate primary antibody diluted in blocking buffer overnight at 4C.
16h
Wash samples with PBS 3 times
5m
Incubate samples with PBS for 5 minutes, repeat 3 times
18m
Incubate with secondary antibody diluted to 1:1000 in blocking buffer for 2h
2h
Mounting
Mounting
30m
30m
Wash samples with PBS 3 times
5m
Incubate samples with PBS for 5 minutes, repeat 3 times
17m
Add 1:100 DAPI diluted in PBS, incubate for 10 minutes
10m
Wash samples with PBS 2 times
Add mounting media, probably PBS in this case or mowiol mixed with 10% dabco