Anti-GFAP antibody [RM1003] - Astrocyte Marker

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GFAP antibody [RM1003] - Astrocyte Marker (AB278054)

Immunohistochemical analysis of paraffin-embedded human cerebrum tissue labeling GFAP with ab278054 at 1/2000 (0.235 μg/ml) dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human cerebrum. The section was incubated with ab278054 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GFAP antibody [RM1003] - Astrocyte Marker (AB278054)

Immunohistochemical analysis of paraffin-embedded rat cerebrum tissue labeling GFAP with ab278054 at 1/2000 (0.235 μg/ml) dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on rat cerebrum. The section was incubated with ab278054 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-GFAP antibody [RM1003] - Astrocyte Marker (AB278054)

Immunofluorescence staining of GFAP using ab278054 in primary rat hippocampal mixed glia, (prepared from P2 rat hippocampal brain area, obtained from Transnetyx Tissue by BrainBits, LLC, cat.no. SDPHP4m), DIV4. The cells were fixed with 100% MeOH (5 min), permeabilized with 0.1% Triton-X-100 (in PBS) for 5 mins and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab278054 at 0.1 μg/ml and ab4674, Anti-GFAP antibody, at 1/1000 dilution. Cells were then incubated with ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150176, Goat Anti-Chicken IgY H&L (Alexa Fluor® 594) preadsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue). Images were acquired with the Perkin Elmer Operetta HCA and a maximum intensity projection of confocal sections is shown. The antibody ab278054 gave comparable results using 4% formaldehyde fixation (10 min).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GFAP antibody [RM1003] - Astrocyte Marker (AB278054)

Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue labeling GFAP with ab278054 at 1/2000 (0.235 μg/ml) dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on mouse cerebrum. The section was incubated with ab278054 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-GFAP antibody [RM1003] - Astrocyte Marker (AB278054)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized mouse cerebrum tissue labeling GFAP with ab278054 at 1/700 (0.938 μg/ml) dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at a 1/1000 dilution. Nuclear counterstain is DAPI. Positive staining on mouse cerebrum.

Secondary antibody only control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) used at a 1/1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10 mM citrate pH 6.0 + 0.05% Tween-20).

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-GFAP antibody [RM1003] - Astrocyte Marker (AB278054)

Flow Cytometry analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized mouse primary brain cells labeling GFAP using ab278054 at a 1/500 dilution (0.1 μg) (Right panel) compared to Rabbit monoclonal IgG (ab172730) (Left panel).

Secondary antibody is Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) used at a 1/2000 dilution.

• IP

Lab

Immunoprecipitation - Anti-GFAP antibody [RM1003] - Astrocyte Marker (AB278054)

GFAP was immunoprecipitated from 0.35 mg mouse brain lysate with ab278054 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab278054 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Mouse brain lysate 10 μg.
Lane 2 : ab278054 IP in mouse brain lysate.
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab278054 in mouse brain lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.
Exposure time : 3 seconds.
The molecular weight observed is consistent with the literature (PMID : 824020; PMID : 2294; PMID : 6340792).

All lanes:

Immunoprecipitation - Anti-GFAP antibody [RM1003] - Astrocyte Marker (ab278054)

Predicted band size: 49 kDa

Observed band size: 40-54 kDa

false

Exposure time: 3s

• WB

Lab

Western blot - Anti-GFAP antibody [RM1003] - Astrocyte Marker (AB278054)

Negative control : Human liver. (PMID : 25975510).
The molecular weight observed is consistent with the literature (PMID : 25975510).

Blocking/Diluting buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-GFAP antibody [RM1003] - Astrocyte Marker (ab278054) at 1/1000 dilution

Lane 1:

Human brain lysate at 20 µg

Lane 2:

Human liver lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 49 kDa

Observed band size: 40-54 kDa

false

Exposure time: 3s

• WB

Lab

Western blot - Anti-GFAP antibody [RM1003] - Astrocyte Marker (AB278054)

The molecular weight observed is consistent with the literature (PMID : 25975510).

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-GFAP antibody [RM1003] - Astrocyte Marker (ab278054) at 1/1000 dilution

Lane 1:

Mouse brain lysate at 20 µg

Lane 2:

Rat brain lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 49 kDa

Observed band size: 40-54 kDa

false

Exposure time: 3s

• WB

Lab

Western blot - Anti-GFAP antibody [RM1003] - Astrocyte Marker (AB278054)

Blocking and Diltuing buffer and concentration : 5% NFDM/TBST

GFAP is expressed primarily by astrocytes in the CNS as astrocytes marker. We have no suitable astrocyte for western blot testing.
U-87 MG and C6 were reported to express low level of GFAP (PMID : 23839947, PMID : 22096544, PMID : 20669222).

NIH/3T3, IMR-32, HeLa and PC-12 were reported as negative cell lines for GFAP (PMID : 824020; PMID : 2294; PMID : 6340792; PMID : 9466565, PMID : 7895062, PMID : 28700643, PMID : 19272755).

No literature was found to support the expression in SH-SY5Y, Neuro-2a and SK-N-BE (2) cell.

All lanes:

Western blot - Anti-GFAP antibody [RM1003] - Astrocyte Marker (ab278054) at 1/1000 dilution

Lane 1:

U-87 MG (Human glioblastoma-astrocytoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

SK-N-BE (2) (Human neuroblastoma neuroblast) whole cell lysate at 20 µg

Lane 4:

IMR-32 (Human lung fibroblast) whole cell lysate at 20 µg

Lane 5:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 6:

Neuro-2a (Mouse neuroblastoma neuroblast) whole cell lysate at 20 µg

Lane 7:

NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 8:

C6 (Rat glial tumor glial cell) whole cell lysate at 20 µg

Lane 9:

PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/20000 dilution

Predicted band size: 49 kDa

Observed band size: 40-54 kDa

false

Exposure time: 180s