Anti-Ly6g antibody [EPR22909-135]

• Flow Cyt

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Flow Cytometry - Anti-Ly6g antibody [EPR22909-135] (AB238132)

Flow cytometric analysis of mouse peripheral blood mononuclear cell (PBMC) cells labeling Ly6g with ab238132 at 1/600 dilution. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Cells were stained with isotype control rabbit monoclonal IgG ab172730 (Left) or ab238132 (Right), then stained with anti-CD11b conjugated to BV421.
Gated on viable cells.

• Flow Cyt

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Flow Cytometry - Anti-Ly6g antibody [EPR22909-135] (AB238132)

Flow cytometric analysis of HEK-293T (Human embryonic kidney epithelial cell) transfected with HA-tagged Ly6c overexpression construct (Left) / HEK-293T transfected with HA-tagged Ly6g overexpression construct (Right) cells, labeling Ly6g with ab238132 at 1/600 dilution. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Ly6c (Left) and Ly6g (Right) overexpression cells were surface stained with ab238132 and anti-HA tag conjugated to Alexa Fluor^®647.

ab238132 does not cross-react with Ly6c.

Gated on viable cells.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-Ly6g antibody [EPR22909-135] (AB238132)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse spleen tissue staining with ab325346 at a 1 : 5000 (0.099 µg/ml) dilution, ab238132 anti-Ly6g used at 1 : 100 (0.29 µg/ml) dilution and ab237721 anti-CD3 used at a 1 : 2000 (0.26 µg/ml) dilution.

Panel A : anti-GPCR GPR97 (green; Opal™520), anti-Ly6g (magenta; Opal™690) and anti-CD3 (yellow; Opal™570) on mouse spleen.
Panel B : anti-GPCR GPR97 staining granulocytes in mouse spleen.
Panel C : anti-Ly6g staining neutrophils in mouse spleen.
Panel D : anti-CD3 staining T lymphocytes in mouse spleen.
Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining : in the order of ab325346, ab238132 and ab237721 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

Nuclear counter stain with DAPI.

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ly6g antibody [EPR22909-135] (AB238132)

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling Ly6g with ab238132 at 1/2000 dilution (0.32 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on neutrophils in mouse spleen (PMID : 21976773) is observed. The section was incubated with ab238132 for 30 mins at RT. The immunostaining staining 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 solution 2) for 20 mins.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ly6g antibody [EPR22909-135] (AB238132)

Immunohistochemical analysis of formalin fixed paraffin embedded mouse spleen labelling Ly6g with ab238132 at a concentration of 2µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a ChromoMap DAB kit and anti-rabbit HQ and anti-HQ HRP detection. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32 mins. ab238132 Anti-Ly6g antibody [EPR22909-135] was incubated for 16 mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ly6g antibody [EPR22909-135] (AB238132)

Immunohistochemical analysis of paraffin-embedded mouse lung tissue labeling Ly6g with ab238132 at 1/2000 dilution (0.32 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on neutrophils in mouse lung (PMID : 22434870) is observed. The section was incubated with ab238132 for 30 mins at RT. The immunostaining staining 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 solution 2) for 20 mins.

• Flow Cyt

Lab

Flow Cytometry - Anti-Ly6g antibody [EPR22909-135] (AB238132)

Flow cytometry staining of C57 BL/6 mouse bone marrow cells with ab238132 (right) or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (left). Cells were incubated for 30 min on ice in 1x PBS containing 10μg/ml anti CD16/CD32 and 10% normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody ab238132 or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (1x 106 in 100 μl at 0.04 μg/ml (1/58750)) for 30min on ice. The cells were simultaneously stained with CD11b.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min on ice

Acquisition of >30000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. Events were gated on viable cells.