Nov 22, 2024
Immunofluorescence of cryopreserved tissue sections
- 1Washington University
Protocol Citation: Carolina Lopez 2024. Immunofluorescence of cryopreserved tissue sections. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk8oxxl5r/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: May 28, 2024
Last Modified: November 22, 2024
Protocol Integer ID: 100749
Abstract
Protocol for immunostaining cryopreserved tissue sections
Materials
| A | B | C | |
| Reagent ID | Vendor | Catalog # | |
| Bovine Serum Albumin (BSA) | Sigma | A3059-50G | |
| Saponin | Sigma | 47036-50G-F | |
| AffiniPure Fab Fragment Anti Mouse IgG (H+L) (Fc Block) | Jackson ImmunoResearch | 115-007-003 | |
| TrueBlack | Biotium | 23014 | |
| ProLong Diamond anti fade mountant | Invitrogen | P36961 | |
| Fluormount-G | Invitrogen | 00-4958-02 | |
| Hoechst 33342 | Invitrogen | H3570 | |
| Triton X-100 | Sigma | T8787-100ML |
PROTOCOL FOR TISSUE IFA - CRYOPRESERVED TISSUE SECTIONS
PROTOCOL FOR TISSUE IFA - CRYOPRESERVED TISSUE SECTIONS
2h 51m
2h 51m
1. Wash slides 2x with PBS (00:03:00 ) to remove OCT.
(Optional) Delimitate a circular area around the tissue slice using a hydrophobic slide marker. All the following incubation steps can be done in 50-100 µL total volume (enough for covering the tissue slice). If using more than one tissue slice per slide and different primary antibody mixes make sure the mixes are well separated.
2. Block/permeabilize with PBS/ 1% BSA/ 0.1% Saponin (or 0.2% Triton X-100 diluted in PBS/1% BSA) + Fc Block (1:200) for 00:40:00 at room temperature.
a) No need for permeabilization if staining just for surface proteins. Remove the permeabilization reagent (Saponin or Triton) from the block mixture accordingly.
b) No need to wash before moving to the next step. Gently tap the slide against the paper towel to remove the blocking solution.
3. Incubate with primary antibody mixes for 01:00:00 at Room temperature - mixes prepared in PBS/ 1% BSA/ 0.1% Saponin (or in PBS/ 1% BSA if not staining intracellular proteins).
a) Incubation with primary antibodies can be done at 4 °C Overnight to improve staining.
4. Wash 3x with PBS (00:03:00 )
5. Incubate with secondary antibodies - prepared in PBS/ 1% BSA/ 0.1% Saponin (or in PBS/ 1% BSA if not staining intracellular proteins) - for 00:40:00 at Room temperature
6. Wash 3x with PBS (00:03:00 )
7. Nuclear staining - Incubate with Hoechst (diluted 1 µL in 10 mL of PBS) for 00:10:00 at Room temperature
8. Wash 3x with PBS (00:03:00 )
9. Background quenching - Incubate with 1x TrueBlack (diluted to 1X in 70% EtOH) for 00:03:00 at Room temperature to reduce tissue autofluorescence.
10. Wash 2x with PBS (00:03:00 ) and once with dH2O (00:03:00 )
11. Mount with coverslips and Prolong Antifade mounting fluid or Fluormount-G.
2h 51m
Protocol references
Annotated by IAC - Updated by Lavinia Gonzalez