For Hypoxia probe staining (flow or microscopy) animals were injected I.V. with 100uL probe 1 hour prior to harvest. Mice were euthanized by cervical dislocation (not CO2).
Harvesting the tissue:
- Remove tissue or tumor carefully, avoid damage, don’t squash it or tear it.
- Place in a LABELED plastic cryostat holder (Sakura Tissue-Tek 4566, Cryomold Intermediate, 15x15x15mm; label with an ethanol-resistant marker). Be sure you have it in the orientation you’re interested in, and that all the tissue is touching or nearly touching the bottom. You will be cutting the tissue on the side touching the bottom. Tumors need to be 5mm or smaller to fit well inside the plastic container. Whole mouse spleens will fit this size container.
- Fill the plastic square with OCT solution (Sakura Tissue-Tek OCT compound 4583) covering the tissue. Do not over-fill the plastic square.
- Put 250-500mL of ethanol in an ice bucket and add enough dry ice to cover the bottom (small chunks). Float the tissue/OCT in an ethanol-dry ice bath (I use toothpicks to keep it from sinking).
The OCT will turn white upon freezing. Leave the tissue in the bath until the OCT is entirely white (usually about 5min).
- Dry the tissue/OCT on dry ice (the ethanol will evaporate).
- Wrap the tissue in foil and store in the –80o freezer.
Cutting the tissue/making slides:
- Call ahead of time for time on the cryostat. It’s usually available, but call the day before to be sure.
- Get the tissues out of the freezer and place on dry ice (put a paper towel inbetween the tissue and the dry ice so they can start warming up a little on your way down).
- When you get to the room, sign in on the sign-up sheet and turn the cryostat on. Make sure things are clean and change the blade if necessary. BE EXTREMELY CAREFUL AROUND THE BLADE. ALWAYS LEAVE THE GUARD ON UNTIL ITS TIME TO CUT.
- Turn the temperature of the cryostat down to –16o for tumors and place two of the tissues to be cut inside to begin warming up. Every tissue has its own optimal cutting temp- if the temp is too high, the tissue will be brittle, too low and it will be mushy. –17 o or –18 o is better for spleens.
- Label the slides (ProbeOn Plus, charged slides, Fisherbrand 15-188-52) with a pencil. I usually cut 2-4 slides per tissue, two tissues per slide. But this depends on how many different stains you will be doing.
- Pop your tissue/OCT out of the plastic holder. Place a bubble of OCT solution on the metal cryostat holder and press onto the rough side of your tissue (the opposite side as the one you want to cut). Allow this to freeze for a couple of minutes.
- Insert the metal holder into the cryostat, remove the guard on the blade and carefully align the blade with the tissue. This takes some messing around with, just be sure not to take a giant chunk out of your tissue.
- Cut through the OCT until you get a full slice of tissue.
- Carefully cut a full slice (should be a full or nearly full square). Allow the tissue to stick to the metal plate, arrange and stretch it flat using the provided instruments, and quickly drop your slide onto the tissue. Quickly remove.
- Once the tissue is on the slide, you should not touch it to anything.
- Fix the tissue in 100% acetone for 5 min. Dry and store in the –80o freezer until staining time.
Staining the slides:
- Bring the slides up to room temp and wipe the back with a Kimwipe (remove excess moisture). Do not touch the tissue.
- Draw a circle around the tissue with a marker (on the back side of the slide). This is to mark where your tissue is on the slide for future reference. Place a dot somewhere off of the tissue also for future reference.
- On the front side of the slide, draw a circle around the tissue using an immunopen (ImmEdge Pen, Vector laboratories, H-4000).
- Block and hydrate your tissue in staining buffer (PBS with 5% FBS). Drop ~200ul on top of the tissue using a glass pipet.
- Place in a dark humidifying chamber (Tupperware with wet paper towel inside) to prevent drying. Incubate 15 minutes.
- Remove the buffer carefully with a glass pipet (vacuum).
- Drop ~75ul of primary stain on each sample (antibody should be diluted in staining buffer and Fc block).
- Incubate 30min in the humidifying chamber.
- Remove primary stain with the glass pipet. Submerge in PBS for a couple minutes. Change the buffer 1x, incubate 5 minutes. Dab dry with Kimwipe.
- Drop ~75ul of secondary stain, incubate in the chamber for 30min.
- Wash by submerging in PBS 5min, change buffer and incubate 20min. Dab dry with Kimwipe (don’t touch the tissue).
- Prepare coverslips by putting one drop of Aquamount (Lerner Laboratories, 13800) where the tissue will be. Quickly drop the slides onto the coverslip (upside down) being careful to avoid air bubbles. Don’t move the coverslip once its made contact with the tissue.
- Dry several hours and seal with nail polish around the edges of the coverslip.
Staining concentrations for tumor sections:
Hypoxia probe antibody: 1:50, directly labeled FITC
CD8 APC: 1:25, pharmigen, directly labeled APC
Anti-LSEC: 1:11, miltenyi, biotinylated, use secondary (Av-Cy3)
Av-Cy3: 1:1000, pharmigen
Taking pictures on the microscope:
- Sign up for scope time online. Again, this is usually available but sign up in advance if possible.
- Turn on the machine is this order: Lambda 10-2 (lamp),both black boxes, manipulator (joy stick), camera (grey switch, lower right), computer. The lamp (the first thing you turn on) has to be on for at least 30 minutes before you can turn it off again).
- Open the software (Slidebook).
- Don’t ever go from oil to dry without cleaning your slide thoroughly.