Anti-TMEM119 antibody [106-6] - Microglial marker

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-TMEM119 antibody [106-6] - Microglial marker (AB210405)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized SH-SY5Y (human neuroblastoma epithelial cell) cells labelling TMEM119 with ab210405 at 1/200 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) antibody at 1/1000 dilution.

Confocal image showing cytoplasmic staining in SH-SY5Y cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue).

Negative control : U-937.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) (ab195889) was used to counterstain tubulin at 1/200 dilution (Magenta).

Secondary antibody only control : Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) at 1/1000 dilution.

• Flow Cyt

Lab

Flow Cytometry - Anti-TMEM119 antibody [106-6] - Microglial marker (AB210405)

Flow cytometry analysis of HEK-293T (human embryonic kidney) transfected with Myc-His tagged mouse TMEM119 expression vector labeling TMEM119 with ab210405 at 1/2000 dilution (0.1μg/mL) (right) compared with isotype control rabbit monoclonal IgG ab172730 (Left). Cells were surface-stained with ab210405, then fixed with 2% PFA for 10 minutes and permeabilised with 0.1% Tween-20 for 30 minutes. Next, they were stained with Alexa Fluor^® 647 conjugated Myc-tag antibody and Alexa Fluor^® 488 conjugated secondary antibody. Only Myc-tag (+) population showed TMEM119 positive staining.

Alexa Fluor^® 488 (ab225497) and R-PE (ab225496) conjugated versions are available for this clone.

• Flow Cyt

Collaborator

Flow Cytometry - Anti-TMEM119 antibody [106-6] - Microglial marker (AB210405)

Flow cytometric analysis of acutely isolated primary mouse microglia (P60 BL6 mouse; wildtype CD11b+CD45lo brain cells) cells labeling TMEM119 with ab210405 at 0.5μg/mL (red) and 0.1μg/mL (blue), compared with TMEM119 KO primary mouse brain cells (black) stained with ab210405 at 0.5μg/mL. Goat anti-Rabbit IgG (Alexa Fluor^®488) at 1/500 dilution was used as the secondary antibody.

No signal was detected on the surface of CD11b+CD45lo brain cells from TMEM119 KO mouse (black) stained with ab210405; whereas in wildtype CD11b+CD45lo brain cells, cell surface staining was observed (red 0.5ug/mL; blue 0.1ug/mL).

The data was provided by Ben Barres' lab (Stanford University). PMID : 26884166.

• WB

Lab

Western blot - Anti-TMEM119 antibody [106-6] - Microglial marker (AB210405)

Western blot : Anti-TMEM119 antibody [106-6] (ab210405) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab210405 was shown to bind specifically to TMEM119. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-TMEM119 antibody [106-6] - Microglial marker (ab210405) at 1/1000 dilution

Lane 1:

HEK-293T overexpressing TMEM119 OE-1160 cell lysate at 20 µg

Lane 2:

HEK-293T cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 40 kDa,43 kDa

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