ICAM1 Rabbit Polyclonal Antibody
cat.: ER1910-98
Product Type: Rabbit polyclonal IgG, primary antibodies
Species reactivity: Human
Applications: WB, IF-Cell, FC
Clonality: Polyclonal
Form: Liquid
Storage condition: Shipped at 4℃. Store at +4℃ short term (1-2 weeks). Store at -20℃ long term.
Storage buffer: PBS (pH7.4), 0.1% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration: 2ug/ul
Purification: Immunogen affinity purified.
Molecular weight: Predicted band size: 60 kDa
Isotype: IgG
Immunogen: Recombinant protein within rat ICAM1 aa 1-517.
Positive control: Mouse spleen tissue lysate, Rat kidney tissue lysate, Rat spleen tissue lysate, C6 cell lysate, RAW264.7 cell lysate, RAW264.7, C6.
Subcellular location: Membrane.
Recommended Dilutions:
  WB
  IF-Cell
  FC
1:5,000-1:10,000
1:1,000
1:2,000
Uniprot #: SwissProt: P13597 Mouse | Q00238 Rat
Alternative names: Antigen identified by monoclonal BB2 BB 2 BB2 CD 54 CD_antigen=CD54 CD54 Cell surface glycoprotein P3.58 Human rhinovirus receptor ICAM 1 ICAM-1 ICAM1 ICAM1_HUMAN intercellular adhesion molecule 1 (CD54), human rhinovirus receptor Intercellular adhesion molecule 1 Major group rhinovirus receptor MALA 2 MALA2 MyD 10 MyD10 P3.58 Surface antigen of activated B cells, BB2
Images
ER1910-98_1.jpg Fig1: Western blot analysis of ICAM1 on different lysates with Rabbit anti-ICAM1 antibody (ER1910-98) at 1/5,000 dilution.

Lane 1: Mouse spleen tissue lysate
Lane 2: Rat kidney tissue lysate
Lane 3: Rat spleen tissue lysate

Lysates/proteins at 15 µg/Lane.

Predicted band size: 60 kDa
Observed band size: 75-120 kDa

Exposure time: 4 seconds; 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 (ER1910-98) at 1/5,000 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.
ER1910-98_2.jpg Fig2: Western blot analysis of ICAM1 on different lysates with Rabbit anti-ICAM1 antibody (ER1910-98) at 1/10,000 dilution.

Lane 1: C6 cell lysate
Lane 2: RAW264.7 cell lysate
Lane 3: RAW264.7 cell lysate treated with deglycosylation

Lysates/proteins at 20 µg/Lane.

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

Exposure time: 20 seconds; 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 (ER1910-98) 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.
ER1910-98_3.jpg Fig3: Immunocytochemistry analysis of RAW264.7 cells labeling ICAM1 with Rabbit anti-ICAM1 antibody (ER1910-98) at 1/1,000 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 (ER1910-98) at 1/1,000 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.
ER1910-98_4.jpg Fig4: Immunocytochemistry analysis of C6 cells labeling ICAM1 with Rabbit anti-ICAM1 antibody (ER1910-98) at 1/1,000 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 (ER1910-98) at 1/1,000 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.
ER1910-98_5.jpg Fig5: Flow cytometric analysis of RAW264.7 cells labeling ICAM1.

Cells were washed twice with cold PBS and resuspend. Then stained with the primary antibody (ER1910-98, 1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). 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).
ER1910-98_6.jpg Fig6: Flow cytometric analysis of C6 cells labeling ICAM1.

Cells were washed twice with cold PBS and resuspend. Then stained with the primary antibody (ER1910-98, 1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). 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).
ER1910-98_7.jpg Fig7: Tissue/cell: rat brain tissue; 4% Paraformaldehyde-fixed and paraffin-embedded;
Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30min; Blocking buffer (normal goat serum,C-0005) at 37℃ for 20 min;
Incubation: Anti-CD54/ICAM-1 Polyclonal Antibody, Unconjugated(bs-0608R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining
ER1910-98_8.jpg Fig8: Blank control: HUVEC cells(blue). Primary Antibody:Rabbit Anti-CD54 antibody(bs-0608R), Dilution: 1μg in 100 μL 1X PBS containing 0.5% BSA; Isotype Control Antibody: Rabbit IgG(orange) ,used under the same conditions ); Secondary Antibody: Goat anti-rabbit IgG-PE(white blue), Dilution: 1:200 in 1 X PBS containing 0.5% BSA.

Protocol
The cells were fixed with 2% paraformaldehyde (10 min) .Primary antibody (bs-0608R, 1μg /1x10^6 cells) were incubated for 30 min on the ice, followed by 1 X PBS containing 0.5% BSA + 1 0% goat serum (15 min) to block non-specific protein-protein interactions. Then the Goat Anti-rabbit IgG/PE antibody was added into the blocking buffer mentioned above to react with the primary antibody at 1/200 dilution for 30 min on ice. Acquisition of 20,000 events was performed.
ER1910-98_9.jpg Fig9: Blank control: mouse thymouses(blue)
Isotype Control Antibody: Rabbit IgG(orange) ;
Secondary Antibody: Goat anti-rabbit IgG-FITC(white blue),
Dilution: 1:100 in 1 X PBS containing 0.5% BSA ;
Primary Antibody Dilution: 1μg in 100 μL 1X PBS containing 0.5% BSA(green).
ER1910-98_10.jpg Fig10: Blank control (blue line): A431 cells(blue).
Primary Antibody (green line): Rabbit Anti-ICAM1/PE-CY7 Conjugated antibody (bs-0608R-PE-CY7)
Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG-PE-CY7 .


Protocol
The cells were fixed with 70% ice-cold methanol overnight at 4℃ . The cells were then incubated in 1 X PBS/2%BSA/10% goat serum to block non-specific protein-protein interactions followed by the antibody for 15 min at room temperature. Cells stained with Primary Antibody for 30 min at room temperature.Acquisition of 20,000 events was performed.
Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.