Anti-CD21+CD35 antibody [RM2062]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CD21+CD35 antibody [RM2062] (AB318999)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Mouse splenocyte cells labelling CD21+CD35 with ab318999 at 1/50 (10.2 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green).

Confocal image showing membranous staining in mouse splenocytes (shown in green). The counterstain was CD45R/B220 which is observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue).
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Anti-human/mouse CD45R/B220 rat monoclonal antibody (Alexa Fluor® 647) was used to counterstain tubulin at 1/100 5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

• Flow Cyt

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Flow Cytometry - Anti-CD21+CD35 antibody [RM2062] (AB318999)

Flow cytometric analysis of Mouse spleen cells labelling CD21+CD35 with ab318999 at 1/500 dilution (0.1 ug)/Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Mouse spleen cell are co-stained with CD19 conjugated PE/Cy7 (780/60BP).
Gated on viable cell.

• Flow Cyt

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Flow Cytometry - Anti-CD21+CD35 antibody [RM2062] (AB318999)

Flow cytometric analysis of Mouse spleen cells labelling CD21+CD35 with ab318999 at 1/500 dilution (0.1 ug)/Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Mouse spleen cell are co-stained with CD3 conjugated Alexa Fluor®647.
Gated on viable cell.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD21+CD35 antibody [RM2062] (AB318999)

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling CD21+CD35 with ab318999 at 1/2000 (0.255 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : no staining on mouse liver.
The section was incubated with ab318999 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

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-CD21+CD35 antibody [RM2062] (AB318999)

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling CD21+CD35 with ab318999 at 1/2000 (0.255 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse spleen.
The section was incubated with ab318999 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

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

• mIHC

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Multiplex immunohistochemistry - Anti-CD21+CD35 antibody [RM2062] (AB318999)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse spleen tissue staining CD21+CD35 with ab318999 at a 1 : 2000 (0.255 ug/ml) dilution, ab245235 anti-CD19 used at 1 : 1000 (0.444 ug/ml) dilution and ab16669 anti-CD3 used at a 1 : 150 (0.06 ug/ml) dilution.

Panel A : merged staining of anti-CD21+CD35 (green; Opal™520), anti-CD19 (magenta; Opal™690) and anti-CD3 (yellow; Opal™570) on mouse spleen.
Panel B : anti-CD21+CD35 staining B lymphocytes in mouse spleen.
Panel C : anti-CD19 staining B lymphocytes 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 ab318999, ab245235 and ab16669 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IP

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Immunoprecipitation - Anti-CD21+CD35 antibody [RM2062] (AB318999)

CD21+CD35 was immunoprecipitated from 0.35 mg Mouse spleen tissue lysate with ab318999 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab318999 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Mouse spleen tissue lysate 10ug
Lane 2 : ab318999 IP in Mouse spleen tissue lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab318999 in mouse spleen tissue lysate

All lanes:

Immunoprecipitation - Anti-CD21+CD35 antibody [RM2062] (ab318999) at 1/30 dilution

All lanes:

Mouse spleen tissue lysate 10ug

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 15s

• WB

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Western blot - Anti-CD21+CD35 antibody [RM2062] (AB318999)

Negative control : EL4(PMID : 10453011)

Low expression : thymus, EL4.IL-2 (PMID : 2966205)

The identity of the lower MW band at approximately 37 kDa (in lane1- 4) is unknown.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

Exposure time : Lane1-4 : 180 seconds, Lane5-6 : 26 seconds

All lanes:

Western blot - Anti-CD21+CD35 antibody [RM2062] (ab318999) at 1/1000 dilution

Lane 1:

WEHI-231 (mouse B cell lymphoma B lymphocyte ) whole cell lysate at 20 µg

Lane 2:

EL4 (mouse lymphoma T lymphocyte) whole cell lysate at 20 µg

Lane 3:

A20 (mouse reticum sarcoma b lymphocyte) whole cell lysate at 20 µg

Lane 4:

EL4.IL-2 (mouse lymphoma T lymphocyte) whole cell lysate at 20 µg

Lane 5:

Mouse spleen tissue lysate at 20 µg

Lane 6:

Mouse thymus tissue 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

Observed band size: 190 kDa,250 kDa,36 kDa

false