TITLE: OHSU SenNet Senescence-Associated Beta-Galactosidase (SA b-Gal) Staining of Carboxymethyl Cellulose (CMC) Embedded Skin Tissue Sections on Polyethylene Naphthalate (PEN) Coated Slides
AUTHORS: Julia R Unsworth, Xianmin Luo, Sheila A. Stewart, Megan K. Ruhland

[DESCRIPTION]
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.  

[STEPS]
SECTION: TISSUE SECTIONING AND EMBEDDING PROCEDURE
1. Collecting the Skin
SECTION: TISSUE SECTIONING AND EMBEDDING PROCEDURE
2. Embedding the Skin in CMC
SECTION: TISSUE SECTIONING AND EMBEDDING PROCEDURE
1.1. From the wet lab take petri dishes for each mouse that you are collecting tissue from and place in a bucket of ice
SECTION: TISSUE SECTIONING AND EMBEDDING PROCEDURE
1.2. Once in mouse room, follow protocol for euthanizing the mice
SECTION: TISSUE SECTIONING AND EMBEDDING PROCEDURE
1.3. Once the mice are euthanized, use an electric razor to shave the backs of each mouse 
SECTION: TISSUE SECTIONING AND EMBEDDING PROCEDURE
1.4. Apply a generous amount of Nair onto the back of each mouse and let sit for ~ 1 min 
SECTION: TISSUE SECTIONING AND EMBEDDING PROCEDURE
1.5. Using a paper towel, carefully wipe the Nair off the mouse’s back and spray with ethanol to clean area
SECTION: TISSUE SECTIONING AND EMBEDDING PROCEDURE
1.6. 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  
SECTION: TISSUE SECTIONING AND EMBEDDING PROCEDURE
1.7. Bring the tissue samples back to the wet lab and prepare an ice bucket of dry ice 
SECTION: TISSUE SECTIONING AND EMBEDDING PROCEDURE
1.8. 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  
SECTION: TISSUE SECTIONING AND EMBEDDING PROCEDURE
2.1. Label two Tissue-Tek Cryomolds per each mouse and pipette Carboxymethyl Cellulose (CMC) media into them until they are halfway full 
SECTION: TISSUE SECTIONING AND EMBEDDING PROCEDURE
2.2. 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
SECTION: TISSUE SECTIONING AND EMBEDDING PROCEDURE
2.3. 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 
SECTION: TISSUE SECTIONING AND EMBEDDING PROCEDURE
2.4. 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
SECTION: TISSUE SECTIONING AND EMBEDDING PROCEDURE
2.5. Allow the cryomolds to harden on dry ice for at least 15 minutes before placing in -80°C freezer for long term storage
SECTION: TISSUE SECTIONING AND EMBEDDING PROCEDURE
3. Tissue Sectioning on the Cryostat
SECTION: TISSUE SECTIONING AND EMBEDDING PROCEDURE
3.1. 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
SECTION: TISSUE SECTIONING AND EMBEDDING PROCEDURE
3.2. 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
SECTION: TISSUE SECTIONING AND EMBEDDING PROCEDURE
3.3. Move slides into -80°C freezer until ready for staining procedure
SECTION: STAINING PROCEDURE
4. Fixation of Slides 
SECTION: STAINING PROCEDURE
4.1. 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  
SECTION: STAINING PROCEDURE
4.2. Wash slides in 2% glycerol in PBS for 2 minutes on shaker at room temperature 
SECTION: STAINING PROCEDURE
5. X-gal Staining
SECTION: STAINING PROCEDURE
5.1. Make staining solution 

 



 

SECTION: STAINING PROCEDURE
5.2. Check the pH of the NaPi stock using a pH meter, it should be at 6.0
SECTION: STAINING PROCEDURE
5.3. Check the pH of the final staining solution, for best results the pH should be at 6.0 
SECTION: STAINING PROCEDURE
5.4. Filter the staining solution using a .45um filter to prevent crystals from forming. Protect from light
SECTION: STAINING PROCEDURE
5.5. Use a Kimwipe to absorb excess PBS. Do not allow the tissue section to dry out
SECTION: STAINING PROCEDURE
5.6. 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
SECTION: STAINING PROCEDURE
5.7. To stop staining reaction, fix again for 10 minutes in fixation solution at room temperature on shaker
SECTION: STAINING PROCEDURE
5.8. Wash slides in 2% glycerol in PBS for 5 minutes on shaker at room temperature
SECTION: STAINING PROCEDURE
6. Nuclear counter stain: Nuclear fast red is a counterstain that targets nucleic acids and helps identify the nucleus of cells
SECTION: STAINING PROCEDURE
6.1. Place slides in Nuclear Fast Red for 10 minutes at room temperature
SECTION: STAINING PROCEDURE
6.2. Rinse slides with tap water 3 times
SECTION: STAINING PROCEDURE
6.3.  Place slides in tap water for 3 minutes on shaker
SECTION: STAINING PROCEDURE
6.4. Let slides air-dry at room temperature
Note: For laser ablation, cover slip and mounting media were not used.