ICAM1 Recombinant Rabbit Monoclonal Antibody [PSH17-38]

Application: IHC-Fr

Species: Mouse

Site: small intestine

Sample: Frozen section

Antibody concentration: 1/1,000

Antigen retrieval: Not required

Immunohistochemical analysis of paraffin-embedded mouse lung tissue with Rabbit anti-ICAM1 antibody (HA723921) at 1/2,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA723921) at 1/2,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Western blot analysis of ICAM1 on different lysates with Rabbit anti-ICAM1 antibody (HA723921) at 1/10,000 dilution.

Lane 1: RAW264.7 cell lysate (20 µg/Lane)
Lane 2: Mouse spleen tissue lysate (30 µg/Lane)
Lane 3: Mouse kidney tissue lysate (30 µg/Lane)

Predicted band size: 60 kDa
Observed band size: 120 kDa

Exposure time: 3 minutes; ECL: K1801;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA723921) at 1/10,000 dilution was used in primary antibody dilution (K1803) at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Immunocytochemistry analysis of RAW264.7 cells labeling ICAM1 with Rabbit anti-ICAM1 antibody (HA723921) at 1/200 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-ICAM1 antibody (HA723921) at 1/200 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (HA601187, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

Flow cytometric analysis of mouse spleen cells labeling ICAM1.

Cells were washed twice with cold PBS and resuspend. Then stained with the primary antibody (HA723921, 1/2,000) (red). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

ICAM1 was immunoprecipitated from 0.2 mg mouse spleen tissue lysate with HA723921 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA723921 at 1/10,000 dilution. HRP Conjugated Anti-Rabbit IgG for IP Nano-secondary antibody at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: Mouse spleen tissue lysate (input)
Lane 2: HA723921 IP in mouse spleen tissue lysate
Lane 3: Rabbit IgG instead of HA723921 in mouse spleen tissue lysate

Blocking/Dilution buffer: primary antibody dilution (K1803)
Exposure time: 59 seconds; ECL: K1801

Application: Immunofluorescence (IF-tissue)

Species: Mouse
Tissue: Lung
Sample: Paraffin-embedded section

Antigen retrieval: Heat-mediated, Tris-EDTA buffer (pH 9.0), 20 minutes at 95℃.

Wash buffer: 1× TBST
Blocking: 10% normal goat serum + 1% Triton X-100 + 0.3 M Glycine in TBST, 30 minutes at room temperature.
Primary antibody: HA723921, 1/500, overnight at 4℃.
Secondary antibody: Goat Anti-Rabbit IgG (iFluor™ 488, HA1121), 1.5 hours at room temperature.