Immunofluorescence staining
For this protocol you will need McCoy cell monolayers grown on glass coverslips in a 24 well tray infected with Chlamydia (see here for details). Make up Saponin buffer (10% FCS, 0.1% Saponin, 0.1% NaAz in PBS).
Fixing protocol
- Remove medium from coverslip and wash with 1ml PBS
- Add 1ml of 4% formaldehyde (in PBS)
- Incubate at room temperature for 15min
- Wash coverslip in 1ml PBS
- Store in 1ml Saponin Buffer at 4°C (incubate for at least 1hr)
Staining protocol
Make up all dilutions with Saponin buffer
- We can supply the following (see here for details): chlamydia genus-specific monoclonal antibody MAb29 (Skilton et al., 2007), C. trachomatis (B-complex) species-specific MAb6ciii (Conlan et al., 1989) and C. pneumonia species-specific MAbP33 (Ni et al., 1994).
- Place coverslip on a tray, cells facing upwards
- Add 80μl primary antibody at required dilution and incubate at room temperature for 1hr
- Wash coverslip with Saponin buffer three times
- Place coverslip on tray facing upwards
- Add 80μl of required Alexa Fluor conjugate (we use AF488 (green) 1:2000, but recommend determining concentration experimentally)
- Incubate at room temperature for 30min
- Wash coverslip with Saponin buffer three times
- Either:
- For phase contrast: Mount coverslips on slides with ~8μl vector shield (containing DAPI)
- Or:
- For confocal microscopy: add 80μl Wheat Germ Agglutinin Alexa Fluor Conjugate (1:200) (WGA AF954 for red)
- Incubate at room temperature for 10 minutes
- Wash coverslip with Saponin buffer three times
- Add 80μl DAPI (1:1000)
- Incubate at room temperature for 10min
- Wash coverslip with PBS buffer three times
- Mount coverslip on slides with ~8μl Mowiol mounting medium