Growth on glass slides

Cover slides are incubated with 1 M NaOH for two hours to clean and sterilise the glass surface and washed 3x with sterile water. Following this washing step, you may coat the cover slides with BSA, ConcanavalinA, poly-lysine. You may also use ready-to-use slides such as LabTek chambers
Seed the cells onto the cover slides.

Fixation

wash the cells on the slide in PBS
Fixed the cells in 4% formaldehyde/PBS for 20 min at RT
permeabilise with 0.5% Tx and 0.5% NP-40


Immunostaining

• wash in PBS for 3 x 5 min
• permeabilise in PTX (PBS + 0,5% Triton X-100) for 1 x 5 min
• block in PBT + 0,5% BSA for 30 min
• incubate in primary antibody solution(PBT + 0,1% BSA + diluted Ab) overnight at 4C or for 2h at RT. Depending on the size of the slide use 30-50 ul. Cover the slide with a piece of parafilm.
• wash in PBT. Dip the slide several times into the wash solution.
• incubate with secondary antibody for 1-2 h RT in PBT + 0,1% BSA + 2. ab)
• wash in PBT
• stain the nuclei with DAPI (1:250) for 3-5 min
• For mounting already prepare a slide with one drop of Aquapolymount
• wash off the DAPI with PBT
• briefly rinse with water to p
• briefly remove all liquid from the coverslide with tissue paper. Touch the paper with a corner of the coverslide. Add the coverslide with the cells down onto the drop of Aquapolymount. Prevent any air bubbles.

Coating of cover slides with Concanavalin A

• use a sterile LabTek chamber
• add ConcanavalinA solution on the glass surface. Make sure that the surface is completely covered.
• incubate for 30 minutes on a shaker at RT
• aspirate the Concanavalin solution. Save it for further use.
• 2x rinse the chamber briefly with deionised water
• remove as much water as possible
• put the open chamber into a laminar flow hood until the completly dry
• seed the cells into the chambers

Concanavalin A solution

1 mg/ml Concanavalin A (Sigma, #L7647)
10 mM Na-phosphate buffer pH 6
10 mM CaCl₂
1 mM MnCl₂
0.01% NaN₃