Immunocytochemistry/ Immunofluorescence abreview for Anti-PCNA antibody - Proliferation Marker

Immunocytochemistry/ Immunofluorescence
Human Cell (MRC5 Sv40 transformed fibroblast)
MRC5 Sv40 transformed fibroblast
paraformaldehyde then methanol - see comment
Yes - 0.2% triton in CSK for 2 minutes PRIOR to fixation - see comments
Blocking step
BSA as blocking agent for 20 minute(s) · Concentration: 5% · Temperature: RT°C

Other product details

Incubation time
1 hour(s) and 0 minute(s) · Diluent: 5% BSA in PBS

Secondary antibody

Non-Abcam antibody was used: alexafluor488 goat anti rabbit (H+L) highly cross
Host species: Goat
Clonality: Polyclonal
Conjugation: Alexa Fluor® 488

Additional data

Additional Notes
I used this to look at chromatin bound PCNA specifically, so the protocol was pre-extraction before fixation:
Cells on glass cover slips were washed in PBSS (regular PBS but with 0.5mM MgCl2 and 0.5mM CaCl2 added) then CSK (10mM PIPES pH7.0, 100mM NaCl, 300mM Sucrose, 3mM MgCl2, 1x roche complete protease inhibitors without EDTA) then extracted with 0.2% triton x-100 (from a 10% stock) in CSK for 2 minutes at room temperature.
Then one careful wash in CSK followed by fixation in 2% PAF in PBSS for 20minutes at RT.
Cells were washed in PBSS then placed in 100% methanol at -20 degreesC for 20minutes then washed twice in PBSS.
Block was 20minutes in 5%BSA in PBSS then antibody was added at 1/2000 dilution in block buffer for 1 hour, 50ul per coverslip.
After three washes in PBSplus 0.1% tween, the secondary was for 45 minutes at 1/1000 in blocking buffer plus DAPI at 0.75ug/ml followed by three washes and mounting in aqua-poly/mount.
Images were acquired on a Leica SP5 upright scanning confocal and processed in Adobe photoshop.

The S phase cells (marked by arrowheads on image) show strong nuclear staining which has classic focal replication patterns. These cells counterstain with pulsed BrdU confirming that this pattern is S phase specific. The non-S phase cells do show some staining which I presume is non-specific. It is strongest in the nucleolar regions and very distinctive so scoring of S vs non-S cells with this antibody is very simple.

Dr. Catherine Green

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Submitted Jan 09 2008

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