Jun 05, 2024
Version 2
Twist2 Snap Frozen Skeletal Muscle IF V.2
This protocol is a draft, published without a DOI.
- Griffen Wakelin1
- 1Dalhousie University
Protocol Citation: Griffen Wakelin 2024. Twist2 Snap Frozen Skeletal Muscle IF. protocols.io https://protocols.io/view/twist2-snap-frozen-skeletal-muscle-if-dexr3fm6Version created by Griffen Wakelin
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: May 18, 2024
Last Modified: June 05, 2024
Protocol Integer ID: 101073
Keywords: if, ihc, twist2, skeletal muscle, muscle
Abstract
This protocol details Twist2 immunofluorescent staining of fresh frozen mouse skeletal muscle.
Image Attribution
Representative Twist2 stain in skeletal muscle. Griffen Wakelin. June 1st, 2024. Dalhousie University.
Protocol materials
Anti-Twist2 antibodyAbcamCatalog #ab66031
Step 7
Bovine Serum AlbuminMerck MilliporeSigma (Sigma-Aldrich)Catalog #A7030
Step 6
Triton X-100Fisher ScientificCatalog #BP151-100
Step 4
ParaformaldehydeMerck MilliporeSigma (Sigma-Aldrich)Catalog #P6148
Step 2
Hydrophobic Barrier PenVector LaboratoriesCatalog #H-4000
Step 1
FluoromountMerck MilliporeSigma (Sigma-Aldrich)Catalog #F4680
Step 11
Alexa Fluor® 488 AffiniPure™ Goat Anti-Rabbit IgG (H L)Jackson ImmunoResearch Laboratories, Inc.Catalog #111-545-003
Step 9
DAPIInvitrogen - Thermo FisherCatalog #D3571
Step 9
Before start
Collect fresh muscle according to institution-specific guidelines and snap freeze in liquid nitrogen-cooled 2-methylbutane. The Twist2 stain does not work on PFA-fixed muscle without antigen retrieval.
Day 1
Day 1
10m
Thaw slides, dry for 30 mins, surround sections with Hydrophobic Barrier PenVector LaboratoriesCatalog #H-4000 , and label
Fix with ice-cold 4 % (v/v) PFA/PBS ParaformaldehydeMerck MilliporeSigma (Sigma-Aldrich)Catalog #P6148 for 00:05:00
5m
Wash three times with 1X PBS for 00:05:00
5m
Permeabilize with 0.2 % (v/v) Triton/PBS Triton X-100Fisher ScientificCatalog #BP151-100 for 00:10:00
10m
Wash three times with 1X PBS for 00:05:00
5m
Block with 5 Mass / % volume BSA/PBS Bovine Serum AlbuminMerck MilliporeSigma (Sigma-Aldrich)Catalog #A7030 for 01:00:00 at Room temperature
1h
Incubate primary Anti-Twist2 antibodyAbcamCatalog #ab66031 1:500 in 1% BSA Overnight at 4 °C
Day 2
Day 2
30m
Wash three times with 1X PBS for 00:05:00
5m
Incubate secondary Alexa Fluor® 488 AffiniPure™ Goat Anti-Rabbit IgG (H+L)Jackson ImmunoResearch Laboratories, Inc.Catalog #111-545-003 1:1000 and DAPIInvitrogen - Thermo FisherCatalog #D3571 1:1000 in 1% BSA for 02:00:00 at Room temperature
2h
Wash three times with 1X PBS for 00:05:00
5m
Mount with aqueous mounting media such as FluoromountMerck MilliporeSigma (Sigma-Aldrich)Catalog #F4680 . Image within 2 days.