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PCNA (PC10) Mouse mAb (Alexa Fluor® 488 Conjugate) #8580
Confocal immunofluorescent analysis of NCI-H460 cells using PCNA (PC10) Mouse mAb (Alexa Fluor® 488 Conjugate) (green), p21 Waf1/Cip1 (12D1) Rabbit mAb (Alexa Fluor® 647 Conjugate) #8587 (blue pseudocolor), and Phospho-Histone H3 (Ser10) (D2C8) XP® Rabbit mAb (Alexa Fluor® 555 Conjugate) #3475 (red).Learn more about how we got this image
Gallery: PCNA (PC10) Mouse mAb (Alexa Fluor® 488 Conjugate) #8580
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
- 20X Phosphate Buffered Saline (PBS): (9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
- Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use.
- Methanol, 100%
- Blocking Buffer (1X PBS / 5% normal goat serum (#5425) / 0.3% Triton™ X-100): To prepare 10 ml: add 0.5 ml normal goat serum and 0.5 ml 20X PBS to 9.0 ml dH2O, mix. While stirring, add 30 µl Triton™ X-100.
- Antibody Dilution Buffer (1X PBS / 1% BSA / 0.3% Triton X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (9998), mix.
- Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).
B. Specimen Preparation - Cultured Cell Lines (IF-IC)
NOTE: Cells should be grown, treated, fixed and stained directly in multiwell plates, chamber slides or on coverslips.
- Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde in 1X PBS.
NOTE: Formaldehyde is toxic, use only in fume hood.
- Allow cells to fix for 15 minutes at room temperature.
- Aspirate fixative, rinse three times in 1X PBS for 5 minutes each.
- Proceed with Immunostaining (Section C).
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
- Methanol Permeabilization Step: Cover cells with ice-cold 100% methanol (use enough to cover completely to a depth of 3–5 mm, DO NOT LET DRY), incubate in methanol for 10 minutes at –20°C, rinse in 1X PBS for 5 minutes.
- Block specimen in Blocking Buffer for 60 minutes.
- While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
- Aspirate blocking solution, apply diluted primary antibody.
- Incubate overnight at 4°C.
- Rinse three times in 1X PBS for 5 minutes each.
- Coverslip slides with Prolong® Gold Antifade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).
- For best results examine specimens immediately using appropriate excitation wavelength. For long term storage, store slides flat at 4°C protected from light.
posted November 2006
revised December 2010
protocol id: 220
NOTE: Prepare solutions with Milli-Q or equivalently purified water.
- 1X Phosphate Buffered Saline (PBS): Dissolve 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.24 g KH2PO4 in 800 ml dH2O. Adjust the pH to 7.4 with HCl and the volume to 1 L. Store at room temperature.
- Absolute Methanol
- Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) in 100 ml 1X PBS. Store at 4°C.
- Permeabilization Buffer: 0.5% Triton X-100, 0.2 µg/ml EDTA and 1% BSA in PBS.
- Recommended Anti-Mouse secondary antibodies::
- Anti-Mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4408
- Anti-Mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 555 Conjugate) #4409
- Anti-Mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 594 Conjugate) #8890
- Anti-Mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4410
- Anti-Mouse IgG (H+L), F(ab')2 Fragment (PE Conjugate) #8887
A. Isolation of Cells and Fixation
- Collect cells by centrifugation and aspirate supernatant.
- Resuspend cells briefly in 100 µl PBS.
- Add 500 µl of permeabilization buffer. Incubate for 15 minutes on ice.
- Add 3 ml of ice-cold 100% methanol and mix on and off for 10 minutes.
- Proceed with staining or store cell at -20°C.
NOTE: Account for isotype matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies. Count cells using a hemocytometer or alternative method.
- Aliquot 0.5–1x106 cells into each assay tube (by volume).
- Add 2–3 ml Incubation Buffer to each tube and rinse by centrifugation. Repeat.
- Resuspend cells in 100 µl Incubation Buffer per assay tube.
- Block in Incubation Buffer for 10 minutes at room temperature.
- Add the fluorochrome-conjugated primary antibody at the appropriate dilution to the assay tubes (see individual antibody datasheet for the appropriate dilution).
- Incubate for 1 hour at room temperature.
- Rinse as before in Incubation Buffer by centrifugation.
- Resuspend cells in 0.5 ml PBS and analyze on flow cytometer.
- Incubate for 30 minutes at room temperature.
- Rinse as before in Incubation Buffer by centrifugation.
- Resuspend cells in 0.5 ml PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to step C1.
C. Optional DNA Stain
- Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
- Incubate for at least 5 minutes at room temperature.
Analyze cells in DNA stain on flow cytometer.
posted April 2012
protocol id: 45
PCNA (PC10) Mouse mAb (Alexa Fluor® 488 Conjugate) detects endogenous levels of total PCNA protein.Species Reactivity: Human, Mouse, Rat, Monkey, Bovine, Pig
Monoclonal antibody is produced by immunizing animals with a recombinant Protein A-PCNA fusion protein obtained from pC2T.
This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 488 fluorescent dye and tested in-house for direct flow cytometric and immunofluorescent analysis in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated PCNA (PC10) Mouse mAb #2586.
Proliferating cell nuclear antigen (PCNA) is a member of the DNA sliding clamp family of proteins that assist in DNA replication (1). Crystal structure data suggests that a PCNA homotrimer ring can encircle and slide along the DNA double helix (2). Multiple proteins involved in DNA replication, DNA repair, and cell cycle control bind to PCNA rather than directly associating with DNA, thus facilitating fast processing of DNA (reviewed in 3). PCNA protein expression is a well-accepted marker of proliferation.
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc. The Alexa Fluor® dye antibody conjugates in this product are sold under license from Life Technologies Corporation for research use only, except for use in combination with DNA microarrays. The Alexa Fluor® dyes (except for Alexa Fluor® 430 dye) are covered by pending and issued patents. Alexa Fluor® is a registered trademark of Molecular Probes, Inc.