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) warmed at 37°C
• Fixing Buffer - 4% paraformaldehyde (PFM) in PBS, two alternative recipes...
  • Recipe 1 requires PFM 97% (dry, Alfa Aesar A11313-22), a 20 mL aliquot of 1xPBS (non-sterile)
  • Recipe 2 requires PFM 32% (liquid, stored in glass ampules), a 21.9 mL aliquot of 1x PBS (non-sterile)
• 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 - Immuno Protein Blocking Solution with Horse Serum (Gentex GTX30974)

PROCEDURE:

Fixation

1. Make Fixing Buffer: Work under the fume hood.
   1. Recipe 1: Add 1.0 g dry 97% PFM 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. Recipe 2: Add 3.1 mL of liquid 32% PFM to the 21.9 mL aliquot of non-sterile 1xPBS in a 50 mL conical tube. Add 25 μL 10M NaOH. Cap and invert to mix.
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. Add 200 μL of Blocking Solution to each well. Incubate for at least one hour at room temp. (can incubate overnight at 4°C if necessary).
2. Dilute primary antibody (according to supplier’s instructions) in Blocking Solution. You will need 15 μL per well.
3. 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).
4. Wash 3 – 4 times/ 5 min. with non-sterile 1xPBS.
5. Dilute secondary antibody and Hoechst (DNA stain) in Blocking Solution (see table). You will need 15 μL per well.
6. 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.
7. Wash 3 – 4 times/ 5 min. with PBS.
8. Image the cells using the proper filter setting on the fluorescent microscope, or store under PBS at 4°C, protected from light.

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 reagents listed for Culture Plates...

• Sterilized (autoclaved) round coverslips
• 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.

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. Incubate at room tempertaure for 5 min.
3. Remove the polylysine solution from each well. Let the slips dry in the biosafety cabinet, uncovered for at least 30 min.
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

• Note: Instead of incubating under parafilm, use the slip-inversion approach.

1. Add 200 μL of Blocking Solution to each well. Incubate for at least one hour at room temp. (can incubate overnight at 4°C if necessary).
2. Dilute primary antibody (according to supplier’s instructions) in Blocking Solution. You will need 15 μL per well.
3. Obtain a fresh 24-well plate. Add 15 μL diluted primary to the center of the bottom of a well. Use forceps to carefully remove a cell-coated coverslip from the fixation/permeabilization plate and INVERT the slip cell-side down onto the droplet of antibody. Incubate at room temp for at least one hour in a humidified box (incubate overnight at 4°C if necessary).
4. Wash 3 – 4 times/ 5 min. with non-sterile 1xPBS. Do not shake/ rock the plate. There is no need to turn the cover slip cell-side up.
5. Dilute secondary antibody and Hoechst (DNA stain) in Blocking Solution (see table). You will need 15 μL per well.
6. Stain the cells with diluted secondary (same way as primary) and incubate at Room Tmp. for one hour. Protect the samples from light; fluorophores are light-sensitive.
7. Wash 3 – 4 times/ 5 min. with PBS. Do not shake/ rock the plate.

Mounting and Storage

1. Place a small drop of Mounting Medium onto a clean rectangular glass slide.
2. Invert a cover slip, cell side down, onto the medium and press gently.
3. Blot-away excess mounting medium by placing a folded Kimwipe onto the slip and pressing straight down with uniform pressure (be careful not to slide the cover slip out of place).
4. Let the slip rest for 30 seconds. Pick up the slide and rinse with a distilled water squirt bottle over a waste container (briefly) and gently blot dry.
5. Slides are best viewed after allowing the mounting medium to set overnight (at Room Tmp., protected from light); but slides may be viewed immediately if handled VERY carefully (cover slip may slide out of place and distort cells).
6. Slides may be stored indefinitely at room temp. (morphology is preserved in the mounting medium).

Dilution Guide

Dilutions of some commonly used secondary antibodies and dyes. Dilutions are empirical values based on recent work in the Haynes lab.

Secondary antibody conjugate or dye	Visible color	Excitation/Emission	Dilution	Volume per 500 μL
Cy2	green	489/506	1:200	2.5 μL
Cy3 (Indocarbocyanine)	green	550/570	1:2000	0.25 μL
Cy5	red	650/670	???	???
Alexafluor 488 (similar to GFP)	cyan-green	495/519	1:1000	0.5 μL
Alexafluor 555	yellow-green	555/565	1:1000	0.5 μL
Alexafluor 594 (similar to mCherry)	red	591/618	1:1000	0.5 μL
Hoechst 33325	blue	345/478	1:1000	0.5 μL

Here is a very useful table of fluorochromes from the Salk Institute: http://flowcyt.salk.edu/fluo.html