Immunofluorescent analysis of 100% methanol fixed C6 (Rat glial tumor cell line ) labeling beta IV Tubulin with ab179509 at 1/500 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution(green).

Cytoplasm staining on C6 cell line is observed.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [EPR16776] -Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG (AlexaFluor®594) preadsorbed (ab150120) at 1/1000 dilution (red).
The negative controls are as follows:-
-ve control 1 : ab179509 at 1/500 dilution followed by ab150120 at 1/1000 dilution.
-ve control 2.: ab7291  at 1/1000 dilution followed by ab150077 at 1/1000 dilution.

All lanes : Anti-beta IV Tubulin antibody [EPR16776] (ab179509) at 1/1000 dilution

Lane 1 : Human fetal liver lysate
Lane 2 : Human fetal heart lysate
Lane 3 : Human fetal kidney lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Developed using the ECL technique.

Predicted band size: 50 kDa
Observed band size: 50 kDa


Exposure time: 30 seconds

Blocking and diluting buffer was 5% NFDM /TBST

All lanes : Anti-beta IV Tubulin antibody [EPR16776] (ab179509) at 1/1000 dilution

Lane 1 : Human cerebellum lysate
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 3 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate
Lane 4 : Neuro-2a (Mouse neuroblastoma cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Developed using the ECL technique.

Predicted band size: 50 kDa
Observed band size: 50 kDa


Exposure time: 30 seconds

Blocking and diluting buffer was 5% NFDM /TBST.

Exposure time - Lane 1: 1 second; Lanes 2,3 and 4: 30 seconds

All lanes : Anti-beta IV Tubulin antibody [EPR16776] (ab179509) at 1/1000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Mouse spleen lysate
Lane 3 : Rat brain lysate
Lane 4 : Rat spleen lysate
Lane 5 : C6 (Rat glial tumor cell line) whole cell lysate
Lane 6 : Raw264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 7 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 8 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Developed using the ECL technique.

Predicted band size: 50 kDa
Observed band size: 50 kDa


Exposure time: 30 seconds

Blocking and diluting buffer was 5% NFDM /TBST.
Exposure time - Lane 1: 1 second; Lane 2: 30 seconds; Lanes 3 and 4: 3 minutes; Lanes 5, 6, 7 and 8: 30 seconds

Immunofluorescent analysis of 100% methanol fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cell line labeling beta IV Tubulin with ab179509 at 1/500 dilution, followed by Goat Anti-Rabbit IgG  (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Cytoplasm staining on HeLa cell line is observed.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [EPR16776] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L  (AlexaFluor®594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows:-
-ve control 1 - ab179509 at 1/500  followed by ab150120 at 1/1000.
-ve control 2. - ab7291 at 1/1000 followed by ab150077  at 1/1000.

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling beta IV Tubulin with ab179509 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm staining on Mouse cerebrum is observed. Counter stained with Hematoxylin.

Negative control: Used PBS instead of primary antibody,secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labeling beta IV Tubulin with ab179509 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm staining on Mouse testis is observed. Counter stained with Hematoxylin.
Negative control: Used PBS instead of primary antibody,secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cell line labeling beta IV Tubulin with ab179509 at 1/150 dilution (red) compared with a Rabbit IgG,monoclonal [EPR16776]- Isotype Control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti Rabbit IgG (Alexa Fluor® 488) at 1/500 dilution was used as the secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde-fixed C6 (Rat glial tumor cell line) labeling beta IV Tubulin with ab179509 at 1/150 (red) compared with a Rabbit IgG,monoclonal [EPR16776] -Isotype control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti Rabbit IgG (Alexa Fluor® 488) at 1/500 dilution was used as the secondary antibody.