Immunohistochemistry (Frozen sections) - active Caspase 3 antibody (ab13847)

ab13847 was used in IHC of frozen sections from a rat brain with a kainite lesion. The non lesionned contralateral site serves as a negative control. The sections were fixed with paraformaldehyde. The tissue was perfused with 4% PFA and embedded in OCT compound and cut on the cryostat. The primary antibody was incubated for 12 hours at a dilution of 1/300. A biotin labelled secondary antibody was used at a dilution of 1/300.

Taken from an Abreview submitted by Sophie Pezet

Immunocytochemistry/ Immunofluorescence - active Caspase 3 antibody (ab13847)

ab13847 at a dilution of 1/500 staining Asynchronous HeLa cells by Immunocytochemistry. The antibody was incubated with the cells for 30 minutes and then detected using a Cy3 conjugated Goat Anti-Rabbit IgG (H+L). This antibody gives a predominantly nuclear focal staining pattern in all interphase nuclei investigated.

This image is courtesy of an Abreview by Kirk McManus submitted on 2 December 2005.

This image is courtesy of an Abreview submitted by Dr Kirk McManus

Immunocytochemistry/ Immunofluorescence - active Caspase 3 antibody (ab13847)

HeLa cells were fixed for 10 minutes at room temperature in 3.7% PFA and permeabilised in 0.1% Triton X-100/PBS then incubated with ab13847 (5µg/ml) for 1 hour at room temperature. The top panel shows control cells treated with DMSO. The bottom panel shows HeLa cells treated with 1 mM staurosporine for 4 hours to induce caspase-3 activation. ab13847 staining is shown in green and counterstaining with DAPI is shown in blue. 100x magnification.

The image shows the staining with ab13847 is very faint in the untreated control cultures,  but  very  bright  after  activation  of capsase-3 by treatment with  the staurosporine. (N.B. in these cultures the nuclei are apoptotic).

Roberto Giambruno, Marilena Ciciarello and Patrizia Lavia

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - active Caspase 3 antibody (ab13847)

ab13847 at 1/1000 staining Xenopus laevis wholemount and sectioned tails by IHC-P. The tissue was formaldehyde fixed and blocked prior to incubation with the antibody for 12 hours. An alkaline phosphatase conjugated goat anti-rabbit antibody was used as the secondary.

This image is courtesy of an Abreview submitted by Dr Michael Levin

Western blot - active Caspase 3 antibody (ab13847)

All lanes : Anti-active Caspase 3 antibody (ab13847) at 1 µg/ml

Lane 1 : Pro-Caspase 3 (inactive) recombinant
Lane 2 : Caspase 3 (active) recombinant

Lysates/proteins at 0.1 µg per lane.

Secondary
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Performed under reducing conditions.

Predicted band size : 17 kDa
Observed band size : 17 kDa

Immunocytochemistry/ Immunofluorescence - active Caspase 3 antibody (ab13847)

ICC/IF image of ab13847 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab13847, 5µg/ml) overnight at +4ºC. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 4% PFA fixed (10 min) Hek293, HepG2 and MCF7 cells at 5µg/ml, and in 100% methanol fixed (5 min) HeLa, Hek293 and MCF7 cells at 5µg/ml.

Flow Cytometry - Anti-active Caspase 3 antibody (ab13847)

ab13847 staining active caspase 3 in Human Jurkat cells by Flow Cytometry. Cells were prepared in a phosphate buffered solution containing 0.1% sodium azide with FBS fixed with paraformaldehyde and permeabilized with Triton X-100 and NP40. The sample was incubated with the primary antibody (1/100 in wash buffer) for 24 hours at 4ºC. A FITC-conjugated Goat anti-rabbit Ig (1/100) was used as the secondary antibody.

Gating Strategy: Isolate cell population from plot of SSC-A / FSA-A

This image is courtesy of an anonymous Abreview

Western blot

All lanes : Anti-active Caspase 3 antibody (ab13847) at 1/500 dilution

Lane 1 : Hela whole cell lysate at 20 µg
Lane 2 : A431 whole cell lysate at 20 µg
Lane 3 : Jurkat whole cell lysate at 20 µg
Lane 4 : Hela whole cell lysate at 20 µg with active Caspase 3 peptide (ab13848) at 1 µg/ml
Lane 5 : A431 whole cell lysate at 20 µg with active Caspase 3 peptide (ab13848) at 1 µg/ml
Lane 6 : Jurkat whole cell lysate at 20 µg with active Caspase 3 peptide (ab13848) at 1 µg/ml
Lane 7 : Recombinant Human caspase 3 (active) recombinant protein at 0.1 µg
Lane 8 : Recombinant Human caspase 3 (active) recombinant protein at 0.1 µg with active Caspase 3 peptide (ab13848) at 1 µg/ml

Secondary
Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP) (ab6721) at 1/5000 dilution
developed using the ECL technique

Predicted band size : 17 kDa

Lanes 1-6: Exposure time 5 min.
Lanes 7-8: Exposure time 30 sec.

ab13847 detects a 17kDa band when tested on recombinant active Caspase 3. This was produced by combining the 17kDa and 12kDa subunits produced separately as recombinant proteins. ab13847 also detects a 17kDa band on lysates from HeLa, A431 and Jurkat cells. These bands can be blocked by the immunising peptide. This strongly suggests that ab13847 is specific for active Caspase 3.