Anti-CD40 antibody [EPR23890-151]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [EPR23890-151] (AB252428)

Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labeling CD40 with ab252428 at 1/500 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on stromal cells of mouse colon is observed. The section was incubated with ab252428 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

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [EPR23890-151] (AB252428)

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling CD40 with ab252428 at 1/500 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Membranous and cytoplasmic staining on mouse spleen (PMID : 25316820) is observed. The section was incubated with ab252428 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.

• IP

Unknown

Immunoprecipitation - Anti-CD40 antibody [EPR23890-151] (AB252428)

CD40 was immunoprecipitated from 0.35 mg RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) treated with 1 ug/ml LPS (Lipopolysaccharide) for 6 hours whole cell lysate with ab252428 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab252428 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : RAW 264.7 treated with 1 ug/ml LPS (Lipopolysaccharide) for 6 hours whole cell lysate 10 ug

Lane 2 : ab252428 IP in RAW 264.7 whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab252428 in RAW 264.7 whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 3 minutes

All lanes:

Immunoprecipitation - Anti-CD40 antibody [EPR23890-151] (ab252428)

Predicted band size: 30 kDa

false

• WB

Lab

Western blot - Anti-CD40 antibody [EPR23890-151] (AB252428)

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

LPS induces the expression of CD40 in Raw 264.7 cells (PMID : 23605514)

Exposure time : Lanes 1-2 : 10 seconds Lane 3 : 48 seconds.

All lanes:

Western blot - Anti-CD40 antibody [EPR23890-151] (ab252428) at 1/1000 dilution

Lane 1:

Untreated RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 2:

RAW 264.7 treated with 1 μg/ml LPS (Lipopolysaccharide) for 6 hours whole cell lysate at 20 µg

Lane 3:

Mouse lymph node tissue lysate at 20 µg

Secondary

Lane 1:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Lanes 2 - 3:

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 1/50000 dilution

Predicted band size: 30 kDa

Observed band size: 40 kDa

false