Haynes:Mamalian IFC

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Immunocytology of Fixed Cells
Karmella Haynes 2013

Culture Plates

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.

REAGENTS:

  • 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

PROCEDURE:

Fixation

  1. 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.
  2. 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.
  3. Take the plate to your bench.
  4. Remove the 1xPBS from the cells.
  5. Work under the fume hood: Add 0.5 mL freshly made Fixing Buffer to each well. Incubate at Room Tmp./ 10 minutes.
  6. Remove the Fixing Buffer. Add 0.5 mL Permeabilization Solution to each well and incubate at room temp. for 10 min.
  7. 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.

Antibody Staining

  1. Block in 250 μL Blocking Solution for at least one hour at Rm. Tmp. (can incubate overnight at 4°C if necessary).
  2. Dilute primary antibody (according to supplier’s instructions) in Blocking Solution'. Add 15 μl 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).
  3. Wash 3 – 4 times/ 5 min. with non-sterile 1xPBS.
  4. Dilute secondary antibody and Hoechst (DNA stain) in Blocking Solution (see table).
  5. 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.
  6. Wash 3 – 4 times/ 5 min. with PBS.


Mounted Slides

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 ones listed for Culture Plates...

  • PLL Solution - 1 mg/mL poly-L-lysine
  • Vinol Mounting Medium - 15% Vinol (Sigma P8136); 33% glycerol; 0.1% sodium azide

Cell Culturing on Glass Slips

  1. Place a single sterile round cover slip into each well in a 24-well dish.
  2. Add 200 uL polylysine solution to each well.
  3. Remove the polylysine solution and wash once with 0.5 mL 1xPBS.
  4. Seed cells at a fairly sparse density (non confluent). Allow the cells to attach to the cover slips (~ 8 hours)

Fixation

  • Follow instructions for Culture Plates.

Antibody Staining

  • Follow instructions for Culture Plates.

Mounting and Storage

  1. Place a small drop of Vinol Mounting Medium onto a clean slide.
  2. Invert a cover slip, cell side down, onto the medium and press gently.
  3. Blot excess mounting medium with a Kimwipe (be careful not to slide the cover slip out of place).
  4. Rinse briefly with distilled water and blot dry.
  5. 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.
  6. Slides may be stored indefinitely at room temp. (morphology is preserved)


Dilution Guide

Dilutions of some commonly used secondary antibodies and dyes.

Secondary antibody conjugate or dye Visible color Excitation/Emission Dilution
Cy5 red ---/--- 1:200
Cy3 green (FITC) ---/--- 1:2000
Alexafluor 488 green (FITC) ---/--- 1:1000
Hoechst 33325 blue 1:1000

Images: For the Nikon scope, images using the 60X objective have a scale of 0.104167 μM per pixel. For the 20 x objective it’s 0.312501 μM/ pixel.