ESO wikiadmin: Created page with "{| class="wikitable" |+ !Materials ! |- | |Coverslips (round, 12 mm) |- | |24-well plate |- | |4% Paraformaldehyde (PFA) in PBS or |- | |ice-cold methanol or acetone (-20°C) in porcelain well plate |} === Procedure === ---- Cells: * One day before seeding the cells: distribute cleaned and sterilized coverslips in a 24-well plate and coat with gelatine-, poly-L-lysine (10 µg/mL), or fibronectin (2 µg/mL) solution in PBS overnight (o/n) at 4°C * Aspirate coating so..."

2025-03-07T09:45:38Z

Created page with "{| class="wikitable" |+ !Materials ! |- | |Coverslips (round, 12 mm) |- | |24-well plate |- | |4% Paraformaldehyde (PFA) in PBS or |- | |ice-cold methanol or acetone (-20°C) in porcelain well plate |} === Procedure === ---- Cells: * One day before seeding the cells: distribute cleaned and sterilized coverslips in a 24-well plate and coat with gelatine-, poly-L-lysine (10 µg/mL), or fibronectin (2 µg/mL) solution in PBS overnight (o/n) at 4°C * Aspirate coating so..."

New page

{| class="wikitable"
|+
!Materials
!
|-
|
|Coverslips (round, 12 mm)
|-
|
|24-well plate
|-
|
|4% Paraformaldehyde (PFA) in PBS or
|-
|
|ice-cold methanol or acetone (-20°C) in porcelain well plate
|}

=== Procedure ===
----

Cells:

* One day before seeding the cells: distribute cleaned and sterilized coverslips in a 24-well plate and coat with gelatine-, poly-L-lysine (10 µg/mL), or fibronectin (2 µg/mL) solution in PBS overnight (o/n) at 4°C
* Aspirate coating solution
* Seed the cells in 0.5-1 mL medium: HEK293T cells: 8x104 cells/well fibroblasts: 3x104 cells/well

Bacteria:

* One day before infection, streak bacteria on plate and incubate at 37°C o/n
* Prepare bacteria for infection according to separate protocol

Infection:

* Before infection: investigate cell distribution/attachment and check for contamination
* Infect the cells for desired time, wash, and fix according to separate protocol

===== Fixation =====

* At the end of the assay, wash cells 2 x with 500 µl cold PBS++ (alternatively, fix cells directly without washing by carefully sublayering 500 µl of cold PFA- fixation solution
* For PFA-fixation add 300µl 4% PFA/well and fix for 5 min at RT
* For methanol or acetone fixation: -transfer washed coverslips from PBS into porcelain well plate on ice and add directly into ~200 µl fixative/well (ice-cold MeOH or acetone). Fix/permeabilize for 10 min on ice. Transfer coverslip back to PBS-containing well in plastic 24-well plate. Make sure not to switch orientation of the coverslip.

Preparation of samples for confocal microscopy - Revision history