Immunostanining
Immunostaining Cardiomyocytes
- From Frozen Tissue
Materials/Solutions
- Antigen Retrieval Solution (0.1M Glycine in ddH2O - pH to 7.4)
- To make 500 mL, add 3.76 g of Glycine to 400 mL of ddH2O. pH to 7.4. Add water up to 500 mL. Store at room temperature.
- To make 500 mL, add 3.76 g of Glycine to 400 mL of ddH2O. pH to 7.4. Add water up to 500 mL. Store at room temperature.
- 4% PFA (in fume hood, or wear mask):
- For a 4% paraformaldehyde solution, add 4 g of EM grade paraformaldehyde to 50 mL of H2O. Add 1 mL of 1 M NaOH and stir gently on a heating block at ~60°C until the paraformaldehyde is dissolved. Add 10 mL of 10X PBS and allow the mixture to cool to room temperature. Adjust the pH to 7.4 with 1 M HCl (~1 mL), then adjust the final volume to 100 mL with H2O. Filter the solution through a 0.45-μm membrane filter to remove any particulate matter. Make the paraformaldehyde solution fresh prior to use, or store in aliquots at −20°C for several months. Avoid repeated freeze/thawing. Stable 1 year at −20°C; 3 months at 4°C
- For a 4% paraformaldehyde solution, add 4 g of EM grade paraformaldehyde to 50 mL of H2O. Add 1 mL of 1 M NaOH and stir gently on a heating block at ~60°C until the paraformaldehyde is dissolved. Add 10 mL of 10X PBS and allow the mixture to cool to room temperature. Adjust the pH to 7.4 with 1 M HCl (~1 mL), then adjust the final volume to 100 mL with H2O. Filter the solution through a 0.45-μm membrane filter to remove any particulate matter. Make the paraformaldehyde solution fresh prior to use, or store in aliquots at −20°C for several months. Avoid repeated freeze/thawing. Stable 1 year at −20°C; 3 months at 4°C
- Blocking Solution (1% BSA,1% Gelatin,1% Tween-20, 0.001% NaN3)
- To make 100 mL, add 1.0 g BSA, 0.1 g Gelatin, 0.001 g NaN3, and lastly add 100 ul Tween-20 – make sure you add Tween-20 last. If BSA does not dissolve at room temp, heat to 37°C. Filter the solution through a 0.45-μm membrane filter to remove any particulate matter. Store at 4°C.
- To make 100 mL, add 1.0 g BSA, 0.1 g Gelatin, 0.001 g NaN3, and lastly add 100 ul Tween-20 – make sure you add Tween-20 last. If BSA does not dissolve at room temp, heat to 37°C. Filter the solution through a 0.45-μm membrane filter to remove any particulate matter. Store at 4°C.
- 0.5% Triton in PBS
- 0.1% Triton in PBS
- Vectashield Mounting Media
Slide Preparation
- Poly-Lysine
- Coat chamber with poly-lysine by smearing with 10-ul pipette tip. Incubate for 30 minutes at room temp. Aspirate poly-lysine off and let air dry for 30 minutes.
- Laminin
- Coat chamber with Laminin by smearing with 10-ul pipette tip. Incubate for 30 minutes at room temp. Aspirate poly-lysine off and let air dry for 30 minutes.
- ECM (MaxGel ECM – Sigma #E0282)
- Coat chamber slide with ECM and smear with 10-ul pipette tip. Let dry for 20 minutes at room temp.
- Gelatin
- 1)Prepare the gelatin-coating solution by dissolving 5 g of gelatin in 1 L of heated, deionized H2O (temperature should not exceed 45 °C).
- 2)After the gelatin has dissolved, add 0.5 g of chromium potassium sulfate dodecahydrate. Chromium potassium sulfate dodecahydrate will positively charge the slides allowing them to attract negatively charged tissue sections.
- 3)Filter this solution and store at 2-8 °C until use. It is recommended that this solution be filtered again immediately before use (adjust to room temperature before filtration).
- 4)Place the histological slides into metal racks.
- 1)Prepare the gelatin-coating solution by dissolving 5 g of gelatin in 1 L of heated, deionized H2O (temperature should not exceed 45 °C).
Note: The slides should be cleaned by washing them in soapy water and rinsing them thoroughly, first in tap water and finally in deionized water.
- 5)Dip the racks containing the slides 3 to 5 times (~5 seconds each) into the gelatin-coating solution.
- 6)Remove the racks containing the slides and let them drain. Blot excess solution from the racks onto filter paper (gently tap the racks against the filter paper for better drainage).
- 7)Place the racks containing the slides on the lab bench and cover them with paper towels to protect them from dust.
- 8)Dry at room temperature for 48 hours.
- 9)Dried slides can be put back into the boxes that they arrived in and stored at room temperature until use. Slides intended for cryostat sections can be stored at -20 °C.
Isolation of Cardiomyocytes
1) Obtain frozen tissue from -80 °C freezer and place in glass vial containing 1 mL of isolation solution supplemented with protease and phosphatase inhibitors at 1:100 dilution.
2) Homogenize in 3-4 one second bursts at 7000 rpm with mechanical homogenizer
3) Filter, spin down at 120xg for 2 minutes and then resuspend in 400 ul of isolation solution.
Immunostaining Protocol
1. Pipette a drop of cells onto pre-coated slide/chamber slide and incubate for 1 hour at room temperature or 37 °C. Try both
2. Rinse cells lightly with PBS. Repeat once.
3. Fix with 1 mL of ice cold methanol (stored at -20 °C) for 1 minutes
4. Remove methanol.
5. Incubate with 4% PFA for 3 minutes
6. Wash cells for 20 minutes at room temp with 1 mL of 0.5% Triton solution
7. Wash cells for 15 minutes at room temp with 1 mL of 0.1% Triton solution.
8. Repeat step 7
9. Incubate cells for 30 minutes in antigen retrieval solution (0.1M Glycine, pH 7.4)
10. Wash cells 3xs with PBS
11. Incubate with blocking solution (1:1 blocking stock, PBS) for 45 minutes.
12. Incubate with Primary Ab in blocking solution (1:1 with PBS) overnight (dilution recommended by company – usually 1:200-1:500) at 4 °C. Make sure to put moist paper towel in the box with the slide to avoid evaporation of solution.
13. Wash cells with PBS 3x
14. Add Secondary Ab (1:500-1:1000) in blocking solution (1:1 with PBS) for 1 hour at room temp
15. Wash with PBS 3x
16. Add 1-2 drops of VectaShield for 30 minutes at room temp (do in dark room). Can secure two sides of coverslip at this point and then secure other two after 30 minutes.
17. Coverslip and seal with Nail polish.
18. Image with confocal microscope.
