Feb 11, 2019
Version 1
Bovine satellite cell Pax7 ICC V.1
 Forked from Bovine satellite cell Pax7 ICC
- 1Tufts University
Protocol Citation: Andrew Stout, Natalie Rubio 2019. Bovine satellite cell Pax7 ICC. protocols.io https://dx.doi.org/10.17504/protocols.io.xzyfp7w
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: February 11, 2019
Last Modified: February 11, 2019
Protocol Integer ID: 20248
Keywords: Staining, Immunocytochemistry
Abstract
Staining primary bovine satellite cells for Pax7, a common marker of satellite cells and myogenic potential. Protocol developed for https://www.thermofisher.com/antibody/product/PAX7-Antibody-Polyclonal/PA5-68506 (Thermo Fisher Pax7 antibody PA5-68506; rabbit IgG anti-Pax7)
Guidelines
For reference general volumes for given well formats are:
- 96-well = 100 uL
- 48-well = 150 uL
- 24-well = 300 uL
- 12-well = 500 uL
- 6-well = 750 uL
** recommended to use a PAP-pen to select a smaller region of 6-well plates after initial fixing / washing, as this will save antibody
Materials
MATERIALS
4% paraformaldehyde/1XPBS solution
Goat-anti-rabbit-Alexafluor 488 Thermo Fisher ScientificCatalog #A11008
PBS
VECTASHIELD® Hardset™ Antifade Mounting MediumCatalog #H-1400
PAX7 Polyclonal AntibodyThermo Fisher ScientificCatalog #PA5-68506
Wash buffer (PBS / 5% goat serum / 0.05% NaAzide)
Permeabilization solution (PBS / 0.5% Triton X-100)
PBST (PBS 1:1000 Tween-20)
Phalloidin 594Thermo Fisher ScientificCatalog #A12381
Fixation and Permeabilization (1 hour)
Fixation and Permeabilization (1 hour)
Aspirate media from cells
add cold 4% PFA to cells (enough to cover cells or scaffolds)
Incubate at room temperature for 30 minutes00:30:00
Wash 3x with room temperature PBS
- NOTE: at this point, can parafilm and leave in the fridge overnight (or up to 1 week) before staining
Aspirate PBS and add cold Permeabilization solution for 15 minutes00:15:00
Wash 3x with cold PBST
Primary Stain (1 hour, overnight incubation)
Primary Stain (1 hour, overnight incubation)
Aspirate PBST and add cold Wash buffer for 45 minutes00:45:00
Note
During soak, can move to step 8
Dilute primary antibodies in wash buffer and keep on ice (protected from light). For given antibody, use the following dilutions:
- anti-Pax7 (1:500)
- Phalloidin-594 (1:100)
note* prepare enough antibody solution for all conditions (a little extra is usually good to make sure there is enough)
After step 7 incubation, wash 3x with cold PBST
Add primary antibody solutions and incubate overnight at 4C (parafilm to avoid evaporation)
Secondary Stain (1.5 hours)
Secondary Stain (1.5 hours)
Wash 3x with cold PBST
Aspirate PBST and add cold Wash buffer for 15 minutes00:15:00
Note
during soak, can move to step 13
Dilute secondary antibodies in wash buffer and keep on ice (protected from light). For given antibody, use the following dilutions:
- 488 goat-anti-rabbit (1:500)
note* prepare enough antibody solution for all conditions (a little extra is usually good to make sure there is enough)
for 3D, when not using DAPI mounting media3 steps
In the case where you're not planning to use a dapi mounting media (ie 3D constructs), prepare a DAPI solution in a suitable blocking buffer (ie Wash Buffer or a BSA-containing buffer), and use that to prepare antibody solutions, instead of plain wash buffer
After step 12 incubation, aspirate Wash buffer from cells, and add secondary antibody solutions. Incubate in the dark at room temperate for 60 minutes 01:00:00
Wash cells 3X with cold PBST, leaving the last wash to soak for 5 minutes 00:05:00
Aspirate PBST, and add DAPI mounting media. Cover with cover-slip, and image after 10 minutes 00:10:00
- Pax7 = green
- Actin cytoskeleton = red
- nuclei = blue