Mar 31, 2025
Immunofluorescence staining
- Karyna Tarasova1,
- Sinan Gültekin2,
- iris.gerner Gerner3,
- Florien Jenner4
- 1Veterinary Medicine University;
- 2Vetmeduni Vienna;
- 3Vetmeduni;
- 4University of Veterinary Medicine Vienna
Protocol Citation: Karyna Tarasova, Sinan Gültekin, iris.gerner Gerner, Florien Jenner 2025. Immunofluorescence staining. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbnpz3gpk/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: March 31, 2025
Last Modified: March 31, 2025
Protocol Integer ID: 125757
Keywords: Senescence, immunofluorescence assay
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
H3K9me3/S10p (R20236, nsJ Bioreagents, San Diego, USA) is an epigenetic modification to the DNA packaging protein Histone H3 involved in regulation of senescence. H3K9me3/S10p was detected by immunofluorescence assays.
Materials
µ-Slide 18 Well polymer bottom slides (81816, ibidi GmBH, Gräfelfing Germany)
primary antibody rabbit anti-sheep Recombinant H3K9me3/S10p (R20236, nsJ Bioreagents, San Diego, USA)
goat anti-rabbit Alexa Fluor 488
Before start
The primary antibody rabbit anti-sheep Recombinant H3K9me3/S10p (R20236) was acquired from nsJ Bioreagents (San Diego, USA).
Immunofluorescence staining procedure
Immunofluorescence staining procedure
The cells were washed three times with warm PBS +/+ (Mg2+/Ca2+) and fixed with 4% formol for 10 min at room temperature.
The fixed cells were washed twice and then blocked with Blocking Buffer I (10% FCS, 1% BSA, 0.1%
Triton X100 in PBS +/+ (Mg2+/Ca2+)) for 1h 30 min at room temperature.
The cells were incubated overnight at 4oC with primary antibody rabbit anti-sheep Recombinant H3K9me3/S10p (R20236, nsJ Bioreagents, San Diego, USA) in Blocking Buffer II (5% FCS, 0.5% BSA, 0.1% Triton X100 in PBS +/+ (Mg2+/Ca2+)).
The cells were washed with warm PBS+/+ (Mg2+/Ca2+) and incubated with goat anti-rabbit Alexa Fluor 488 1:600 for 1 h in the dark.
The cells were washed three times. VectaShield mounting medium with DAPI (50011, ibidi GmBH, Gräfelfing Germany) was used and the slides were stored at 4 °C.
Microscope settings and Image acquisition
Microscope settings and Image acquisition
For comparison purposes, microscope settings and image acquisition parameters were kept unaltered throughout parallel documentation procedures. Negative controls were performed in the absence of the primary antibodies.
Observations were made using a Zeiss Observer. Six independent images were acquired per condition.
Computed analysis of foci
Computed analysis of foci
The obtained six independent images were exported to Image J 1.37c software (NIH, Bethesda, MD, USA) for quantification analysis.
Regions of interest (ROIs) outlining complete individual nucleus were done automatically. Following assignment the DAPI channel was thresholded using the Otsu method (Otsu N (1979) Threshold Selection Method from Gray-Level Histograms) and only foci points within the ROI mask were analysed.
The computed analysis of the six individual images was expressed as number of foci present normalized by number of nucleus for each experimental condition and by percentage of area occupied within the nucleus by the foci.