Immunocytology of Fixed Cells
Karmella Haynes 2013
This procedure allows you to fix and stain cells in a 24-well culture plate without mounting slides. Image quality and magnification is limited (10x - 20x), but processing larger sample numbers is easier.
- Adherent cells cultured in a 24-well plate
- Sterile 1xPBS (keep in the TC room)
- Paraformaldehyde 97% (dry) - Alfa Aesar A11313-22
- 20 mL aliquot of non-sterile 1xPBS in a 50 mL conical; for Fixing Buffer
- 10M NaOH
- 50 mL aliquot of non-sterile 1xPBS for washes (keep at your bench)
- Permeabilization Solution - Cold methanol or 0.5% Triton-X100 in PBS
- Note: Different antibodies work better with different permeabilization. Try both and see which works best for your specific antibody.
- Blocking Solution - 5% Normal Horse Serum in PBS
- Make Fixing Buffer: Work under the fume hood. Add 1.0 g dry 97% Paraformaldehyde to the 20 mL aliquot of non-sterile 1xPBS in a 50 mL conical tube. Add 25 μL 10M NaOH. Cap the tube, seal with parafilm, and heat at 60°C with occasional shaking until the paraformaldehyde dissolves. Cool the solution to room temperature (or chill in the refrigerator) before use.
- Do the following in the TC room in the biosafety cabinet: Remove medium from the cells. Add 0.5 mL warm Sterile 1x PBS to each well.
- Take the plate to your bench.
- Remove the 1xPBS from the cells.
- Work under the fume hood: Add 0.5 mL freshly made Fixing Buffer to each well. Incubate at Room Tmp./ 10 minutes.
- Remove the Fixing Buffer. Add 0.5 mL Permeabilization Solution to each well and incubate at room temp. for 10 min.
- Remove Permeabilization Solution and rinse cells with 0.5 mL non-sterile 1xPBS for washes.
Note: This is a stopping point. You can place a lid on the plate, seal it with parafilm, and store the cells +1xPBS at 4°C for several days. Or, proceed immediately to Antibody Staining.
- Add 250 μL of Blocking Solution to each well. Incubate for at least one hour at room temp. (can incubate overnight at 4°C if necessary).
- Dilute primary antibody (according to supplier’s instructions) in Blocking Solution. You will need 15 μL per well.
- Add 15 μL diluted primary on top of the cells and cover with a 1 cm2 piece of parafilm. Incubate at room temp. for at least one hour in a humidified box (incubate overnight at 4°C if necessary).
- Wash 3 – 4 times/ 5 min. with non-sterile 1xPBS.
- Dilute secondary antibody and Hoechst (DNA stain) in Blocking Solution (see table). You will need 15 μL per well.
- Add diluted secondary to samples (same way as primary) and incubate at Room Tmp. for one hour. Protect the samples from light; fluorophores are light-sensitive.
- Wash 3 – 4 times/ 5 min. with PBS.
This procedure is for mounting slides. The process is more involved, but image quality is greater and cells can be imaged under higher magnification (40x oil objective)
REAGENTS: In addition to the reagents listed for Culture Plates...
- PLL Solution - 0.01% Poly-L-lysine solution (Sigma-Aldrich P4707)
- Mounting Medium - Polyvinyl alcohol mounting medium with NPG (Sigma-Aldrich 10979)
Cell Culturing on Glass Cover Slips
Perform all of the following steps in a biosafety cabinet in the TC room.
- Place a single sterile round cover slip into each well in a 24-well dish.
- Add 200 uL polylysine solution to each well. Incubate at room tempertaure for 5 min.
- Remove the polylysine solution from each well. Let the slips dry in the biosafety cabinet, uncovered for at least 30 min.
- Seed cells at a fairly sparse density (non confluent). Allow the cells to attach to the cover slips (~ 8 hours)
- Follow instructions for Culture Plates.
- Follow instructions for Culture Plates.
Mounting and Storage
- Place a small drop of Mounting Medium onto a clean rectangular glass slide.
- Invert a cover slip, cell side down, onto the medium and press gently.
- Blot excess mounting medium with a Kimwipe (be careful not to slide the cover slip out of place).
- Rinse briefly with distilled water and blot dry.
- Slides are best viewed after allowing the mounting medium to set overnight (at Room Tmp., protected from light); slides may be viewed immediately if handled carefully.
- Slides may be stored indefinitely at room temp. (morphology is preserved)
Dilutions of some commonly used secondary antibodies and dyes.
|Secondary antibody conjugate or dye||Visible color||Excitation/Emission||Dilution||Volume per 500 μL|
|Cy3||green (FITC)||---/---||1:2000||0.25 μL|
|Alexafluor 488||green (FITC)||---/---||1:1000||0.5 μL|
|Hoechst 33325||blue||---/---||1:1000||0.5 μL|