Mar 10, 2025
DIO-SPOTlight Immunohistochemistry v.250227
- Matt Oliver1,2
- 1Calakos Lab;
- 2Duke University
- Matt Oliver: Adapted from immunohistochemistry for p-eIF2a and eIF2a, v.230804;
Protocol Citation: Matt Oliver 2025. DIO-SPOTlight Immunohistochemistry v.250227. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7mo38gwz/v1
Manuscript citation:
Oliver, M. L., Caffall, Z. F., Eatman, C. B., Faw, T. D., & Calakos, N. (2024). DIO-SPOTlight Transgenic Mouse to Functionally Monitor Protein Synthesis Regulated by the Integrated Stress Response. BioRxiv, 2024.10.14.618312. https://doi.org/10.1101/2024.10.14.618312
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 27, 2025
Last Modified: March 10, 2025
Protocol Integer ID: 123531
Keywords: ASAPCRN, DIO-SPOTlight IHC, spotlight immunohistochemistry, immunofluorescent labeling in pfa perfusion, using immunofluorescent labeling, spotlight image processing, immunofluorescent, pfa perfusion, spotlight
Funders Acknowledgements:
ASAP
Grant ID: ASAP-020607
Abstract
Abstract
This protocol is to label several targets using immunofluorescent labeling in PFA perfusion-fixed, sagittal 40-µm thick slices, post-mortem mouse brain tissue.
Note: All steps using an orbital shaker were performed at low speed, gently shaking.
Partner protocols: DIO-SPOTlight Confocal Imaging Acquistion v.250227 & DIO-SPOTlight
Image Processing and Analysis v.250227
Troubleshooting
Pre-staining tissue processing, washes, and blocking
Transfer 2-3 desired brain slices per well into a 24 well plate in PBS.
Quench remaining free aldehydes by exchanging PBS for 0.1M glycine in 1x PBS. Incubate at room temperature (RT) for 30 minutes on an orbital shaker.
Perform alkaline antigen retrieval. Exchange quenching buffer for 1% NaOH in 1x PBS for 20 minutes at RT on an orbital shaker.
Note: Make alkaline retrieval buffer fresh. Handle carefully.
Wash and permeabilize samples. Remove alkaline retrieval buffer and Wash 3x for 10 minutes at RT while on an orbital shaker in PBS-T (1x PBS with 0.3% Triton X-100).
Block samples in blocking buffer. Exchange wash buffer for blocking buffer (10% normal donkey serum (NDS) + 1% BSA (w/v) in PBS-T). Incubate samples for 1 hour at RT while on an orbital shaker.
Antibody incubations
Prepare primary antibodies in dilution buffer (5% normal donkey serum (NDS) + 1% BSA (w/v) in PBS-T).
Anti-RFP (rabbit, 1:1000, Rockland Antibodies and Assays, cat no. 600-401-379)
Anti-GFP (chicken, 1:1000, Abcam, cat no. AB13970)
Anti-ChAT (goat, 1:200, Millipore, cat no. AB144P)
Exchange blocking buffer for dilution buffer containing primary antibodies. Incubate at 4C overnight while on an orbital shaker.
The following day, remove primary antibodies and wash samples 3x for 10 minutes each at RT in PBS-T wash buffer (1x PBS with 0.3% Triton X-100) while on an orbital shaker.
Prepare secondary antibodies in dilution buffer (5% normal donkey serum (NDS) + 1% BSA (w/v) in PBS-T).
Alexa Fluor 488 (Donkey-anti-Chicken) 1:500
Alexa Fluor 546 (Donkey-anti-Rabbit) 1:500
Alexa Fluor 647 (Donkey-anti-Goat) 1:500
Exchange wash buffer for dilution buffer containing secondary antibodies and incubate at RT while on an orbital shaker for 1 hour.
Label nuclei, final washes, and slide mounting
Prepare Hoechst 33342 (10ug/ml) in PBS-T wash buffer 1x PBS with 0.3% Triton X-100).
Exchange dilution buffer containing secondary antibodies for Hoechst 33342 in PBS-T and incubate for 10 minutes while on an orbital shaker at RT.
Wash 2 additional time in PBS-T 10 mins RT while on an orbital shaker.
Exchange PBS-T for PBS
Mount slices on microscope slides and seal with Vectashield Vibrance Mounting Media (cat no. H-1700).
Allow to dry overnight in the dark at RT and then store in fridge.