Pericyte Antibody Sampler Kit
cat.: HAK21124
Product Type: Antibody Sampler Kit
Species reactivity: Human
Applications: WB
Form: Liquid
Storage condition: Store at +4℃ after thawing. Aliquot store at -20℃. Avoid repeated freeze / thaw cycles.
Storage buffer: PBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Uniprot #: SwissProt: Q6UVK1 Human | P43121 Human | Q8R2Y2 Mouse | Q9EPF2 Rat | P62736 Human | P62737 Mouse | P62738 Rat | P05622 Mouse | P09619 Human | Q05030 Rat | P15144 Human | P97449 Mouse | P17661 Human | P31001 Mouse | P48675 Rat | P16284 Human
Images
HAK21124_1.jpg Fig1: Western blot analysis of NG2 on different lysates with Rabbit anti-NG2 antibody (HA723167) at 1/10,000 dilution.

Lane 1: A375 cell lysate
Lane 2: SK-MEL-28 cell lysate
Lane 3: MCF7 cell lysate (negative)
Lane 4: HeLa cell lysate
Lane 5: PANC-1 cell lysate
Lane 6: MDA-MB-231 cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 251 kDa
Observed band size: 330 kDa

Exposure time: Lane 1-2: 6 seconds; Lane 3-6: 59 seconds; ECL: K1801;

3-8% 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 (HA723167) at 1/10,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
HAK21124_2.jpg Fig2: Immunocytochemistry analysis of A375 (positive) and SK-Br-3 (negative) labeling NG2 with Rabbit anti-NG2 antibody (HA723167) at 1/100 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-NG2 antibody (HA723167) at 1/100 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.
HAK21124_3.jpg Fig3: Flow cytometric analysis of SK-Br-3 (left, negative) and A375 (right, positive) cells labeling NG2.

Cells were fixed and permeabilized. Then stained with the primary antibody (HA723167, 1/1,000) (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).
HAK21124_4.jpg Fig4: Western blot analysis of alpha smooth muscle Actin on different lysates with Rabbit anti-alpha smooth muscle Actin antibody (ET1607-53) at 1/20,000 dilution and competitor's antibody at 1/2,000 dilution.

Lane 1: Saos-2 cell lysate (15 µg/Lane)
Lane 2: A431 cell lysate (15 µg/Lane)
Lane 3: NIH/3T3 cell lysate (15 µg/Lane)
Lane 4: C2C12 cell lysate (15 µg/Lane)
Lane 5: Neuro-2a cell lysate (15 µg/Lane)
Lane 6: Mouse skin tissue lysate (30 µg/Lane)
Lane 7: Rat skin tissue lysate (30 µg/Lane)

Predicted band size: 42 kDa
Observed band size: 42 kDa

Exposure time: 46 seconds

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 (ET1607-53) at 1/20,000 dilution and competitor's antibody at 1/2,000 dilution were used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:50,000 dilution was used for 1 hour at room temperature.
HAK21124_5.jpg Fig5: Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue with Rabbit anti-alpha smooth muscle Actin antibody (ET1607-53) at 1/50,000 dilution and competitor's antibody at 1/20,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 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 (ET1607-53) at 1/50,000 dilution and competitor's antibody at 1/20,000 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.
HAK21124_6.jpg Fig6: Western blot analysis of alpha smooth muscle Actin on different lysates with Rabbit anti-alpha smooth muscle Actin antibody (ET1607-53) at 1/5,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: A431 cell lysate
Lane 3: A549 cell lysate
Lane 4: NIH/3T3 cell lysate
Lane 5: C2C12 cell lysate
Lane 6: L6 cell lysate
Lane 7: Mouse heart tissue lysate
Lane 8: Mouse skin tissue lysate
Lane 9: Rat heart tissue lysate
Lane 10: Rat skin tissue lysate
Lane 11: Rat smooth muscle tissue lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 42 kDa
Observed band size: 42 kDa

Exposure time: 5 seconds;

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 (ET1607-53) at 1/5,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:100,000 dilution was used for 1 hour at room temperature.
HAK21124_7.jpg Fig7: Immunofluorescence analysis of paraffin-embedded human kidney tissue labeling alpha smooth muscle Actin (ET1607-53).

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 10% negative goat serum for 1 hour at room temperature, washed with PBS. And then probed with the primary antibody alpha smooth muscle Actin (ET1607-53, red) at 1/1,000 dilution overnight at 4 ℃, washed with PBS.

iFluor™ 647 conjugate-Goat anti-Rabbit IgG (HA1123) was used as the secondary antibody at 1/1,000 dilution. DAPI was used as nuclear counterstain.
HAK21124_8.jpg Fig8: Fluorescence multiplex immunohistochemical analysis of the human pancreatic carcinoma (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-CD31 (M1511-8, green), anti-α-SMA (ET1607-53, red) and anti-FAP (ET1704-23, yellow) on human pancreatic carcinoma. Panel B: anti- CD31 stained on the endothelial cells. Panel C: anti-α-SMA stained on cancer-associated fibroblasts and smooth muscle cells. Panel D: anti-FAP stained on the cancer-associated fibroblasts. HRP Conjugated UltraPolymer Goat Polyclonal Antibody HA1119/HA1120 was used as a secondary antibody. The immunostaining was performed with the Sequential Immuno-staining Kit (IRISKit™MH010101, www.luminiris.cn). The section was incubated in three rounds of staining: in the order of M1511-8 (1/5000 dilution), ET1704-23 (1/1000 dilution), and ET1607-53 (1/3000 dilution) for 20 mins at room temperature. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 30 mins at 95℃. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Nikon ECLIPSE Ni-E microscope.
HAK21124_9.jpg Fig9: Immunofluorescence analysis of paraffin-embedded human stomach cancer tissue labeling alpha smooth muscle Actin (ET1607-53) and ALDH2 (M1509-1).

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 10% negative goat serum for 1 hour at room temperature, washed with PBS. And then probed with the primary antibody alpha smooth muscle Actin (ET1607-53, red) at 1/1,000 dilution and ALDH2 (M1509-1, green) at 1/100 dilution overnight at 4 ℃, washed with PBS. iFluor™ 488 conjugate-Goat anti-Mouse IgG (HA1125) and iFluor™ 647 conjugate-Goat anti-Rabbit IgG (HA1123) was used as the secondary antibody at 1/1,000 dilution. DAPI was used as nuclear counterstain.
HAK21124_10.jpg Fig10: Immunocytochemistry analysis of HepG2 (positive) and A431 (negative) labeling alpha smooth muscle Actin with Rabbit anti-alpha smooth muscle Actin antibody (ET1607-53) at 1/2,000 dilution.

Cells were fixed in 100% precooled methanol for 5 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-alpha smooth muscle Actin antibody (ET1607-53) at 1/2,000 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.
HAK21124_11.jpg Fig11: Western blot analysis of PDGF Receptor beta on different lysates with Rabbit anti-PDGF Receptor beta antibody (ET1605-20) at 1/1,000 dilution.

Lane 1: SH-SY5Y cell lysate
Lane 2: C6 cell lysate
Lane 3: Mouse brain tissue lysate
Lane 4: Rat brain tissue lysate

Lysates/proteins at 20 (cell) or 40 (tissue) µg/Lane.

Predicted band size: 150/200 kDa
Observed band size: 190 kDa

Exposure time: 6 seconds;

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 (ET1605-20) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
HAK21124_12.jpg Fig12: CD31 was immunoprecipitated in 0.2mg THP-1 cell lysate with ET1608-48 at 2 µg/25 µl agarose. Western blot was performed from the immunoprecipitate using ET1608-48 at 1/1,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: THP-1 cell lysate (input)
Lane 2: Rabbit IgG instead of ET1608-48 in THP-1 cell lysate
Lane 3: ET1608-48 IP in THP-1 cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 5 seconds
HAK21124_13.jpg Fig13: Immunocytochemistry analysis of C2C12 cells labeling Desmin with Rabbit anti-Desmin antibody (ET1606-30) at 1/500 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-Desmin antibody (ET1606-30) at 1/500 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.
HAK21124_14.jpg Fig14: Immunohistochemical analysis of paraffin-embedded rat heart tissue with Rabbit anti-Desmin antibody (ET1606-30) at 1/1,000 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 (ET1606-30) at 1/1,000 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.
Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.