Jul 31, 2024
OHSU SenNet Senescence-Associated Beta-Galactosidase (SA b-Gal) Staining of Carboxymethyl Cellulose (CMC) Embedded Skin Tissue Sections on Polyethylene Naphthalate (PEN) Coated Slides
- Julia R Unsworth1,
- Xianmin Luo2,
- Sheila A. Stewart2,3,4,
- Megan K. Ruhland1,5
- 1Cell, Developmental and Cancer Biology Department, Oregon Health & Science University;
- 2Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri;
- 3Siteman Cancer Center, Washington University School of Medicine, St. Louis, Missouri Department of Medicine, Washington University School of Medicine, St. Louis, Missouri;
- 4ICCE Institute, Washington University School of Medicine, St. Louis, Missouri;
- 5Dermatology Department, School of Medicine, Oregon Health & Science University
- Cellular Senescence Network (SenNet) Method Development Community
Protocol Citation: Julia R Unsworth, Xianmin Luo, Sheila A. Stewart, Megan K. Ruhland 2024. OHSU SenNet Senescence-Associated Beta-Galactosidase (SA b-Gal) Staining of Carboxymethyl Cellulose (CMC) Embedded Skin Tissue Sections on Polyethylene Naphthalate (PEN) Coated Slides. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbzq7ygpk/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: July 31, 2024
Last Modified: July 31, 2024
Protocol Integer ID: 104333
Funders Acknowledgements:
Spatially-resolved proteome mapping of senescent cells and their tissue microenvironment at single-cell resolution
Grant ID: UG3CA275697
Disclaimer
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Abstract
Senescence-associated beta-galactosidase staining is a common staining technique to detect senescent cells. This protocol describes SA b-Gal staining of mouse skin tissue sections from CMC embedded blocks onto PEN slides which enable the combined use of laser capture microdissection (LCM) and UV laser cutting for downstream mass spectrometry analysis.
TISSUE SECTIONING AND EMBEDDING PROCEDURE
TISSUE SECTIONING AND EMBEDDING PROCEDURE
Collecting the Skin
From the wet lab take petri dishes for each mouse that you are collecting tissue from and place in a bucket of ice
Once in mouse room, follow protocol for euthanizing the mice
Once the mice are euthanized, use an electric razor to shave the backs of each mouse
Apply a generous amount of Nair onto the back of each mouse and let sit for ~ 1 min
Using a paper towel, carefully wipe the Nair off the mouse’s back and spray with ethanol to clean area
Using scissors and a pair of tweezers, cut a rectangle shape of the dorsal skin (from upper shoulders down to lower hips) and place epithelial side up onto the appropriately labeled petri dish
Bring the tissue samples back to the wet lab and prepare an ice bucket of dry ice
Using a clean razor blade, cut off the uneven edges of each tissue segment and then cut into 6-8 smaller rectangle pieces that are all roughly the same size
Embedding the Skin in CMC
Label two Tissue-Tek Cryomolds per each mouse and pipette Carboxymethyl Cellulose (CMC) media into them until they are halfway full
Using tweezers, carefully pick up the cut sections of tissue and place them vertically in the CMC media, making sure they are all oriented the same direction. Place 3-4 tissue pieces in each cryomold
Add more CMC media once tissue pieces are in the cryomold.
Note: Try to have the tissue pieces not sink towards the bottom, and have them all relatively on the same plane
Once tissue sections are vertical, place the cryomolds onto dry ice to harden the CMC media. Add enough CMC media to where you do not see the tissue sections
Allow the cryomolds to harden on dry ice for at least 15 minutes before placing in -80°C freezer for long term storage
Tissue Sectioning on the Cryostat
Take CMC embedded tissue sections out of -80°C freezer, remove block from the cryomold and mount onto pedestal in the Cryostat chamber using a small amount of OCT media to adhere it. Allow to set for ~15-30min
Note: Cryostat should be set at -20°C
Cut tissue sections at 10 μm thickness in the cryostat at -20°C and place onto Polyethylene Naphthalate (PEN) slides
Note: Make sure that the tissue is placed onto the membrane bound side of the slid
Move slides into -80°C freezer until ready for staining procedure
STAINING PROCEDURE
STAINING PROCEDURE
Fixation of Slides
Take tissue slides from -80°C freezer and immediately place in fixation solution for 10 minutes at 4°C on shaker
Fixation Solution
0.2% glutaraldehyde
2% glycerol
in PBS
Wash slides in 2% glycerol in PBS for 2 minutes on shaker at room temperature
X-gal Staining
Make staining solution
| Actual Con. | Stocks | Amount needed for 5ml | |
| 1mg/ml X-gal | 40mg/ml | 125µl | |
| 150mM NaCl | 5M | 150µl | |
| 2mM MgCl2 | 1M | 10µl | |
| 5mM K3Fe(CN)6 | 100mM | 250µl (protect from light) | |
| 5mM K4Fe(CN)6 | 100mM | 250µl (protect from light) | |
| 40mM NaPi pH 6.0 | 0.1M | 2 ml | |
| H2O | 2.217 ml |
* K3Fe(CN)6 and K4Fe(CN)6 should be protected from light
To make up 0.1M NaPi stock (remake every 2 months):
| Actual Con. | Stocks | Amount needed for 10ml | |
| Na2HPO4 | 1M | 125µl | |
| NaH2PO4 | 1M | 880µl | |
| H2O | 8.995 ml |
Check the pH of the NaPi stock using a pH meter, it should be at 6.0
Check the pH of the final staining solution, for best results the pH should be at 6.0
Filter the staining solution using a .45um filter to prevent crystals from forming. Protect from light
Use a Kimwipe to absorb excess PBS. Do not allow the tissue section to dry out
Using a PAP pen, draw a square around the tissue and place ~200ml of staining solution onto tissue and incubate for 6-24 hours at 37°C in a humidified chamber/slide holder
Note: The timing depends on the negative (non-senescent) control tissue, you will
need to adjust the timing based on what is appropriate for your sample
To stop staining reaction, fix again for 10 minutes in fixation solution at room temperature on shaker
Wash slides in 2% glycerol in PBS for 5 minutes on shaker at room temperature
Nuclear counter stain: Nuclear fast red is a counterstain that targets nucleic acids and helps identify the nucleus of cells
Place slides in Nuclear Fast Red for 10 minutes at room temperature
Rinse slides with tap water 3 times
Place slides in tap water for 3 minutes on shaker
Let slides air-dry at room temperature
Note: For laser ablation, cover slip and mounting media were not used.