p16INK4a Recombinant Mouse Monoclonal Antibody [PD01-16]

Immunohistochemical analysis of paraffin-embedded human tonsil tissue with Mouse anti-p16INK4a antibody (HA601131) at 1/500 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) 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 (HA601131) at 1/500 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.

Application: Immunohistochemistry (IHC-P)

Species: Human
Tissue: Cervix poorly differentiated adenocarcinoma
Sample: Paraffin-embedded section

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

Wash buffer: 1× TBST
Endogenous peroxidase blocking: 3% H₂O₂, 10 minutes at room temperature.
Blocking: 1% BSA + 10% normal goat serum, 10 minutes at room temperature.
Primary antibody: HA601131, 1/500, 1 hour at room temperature.
Secondary antibody: HA1120, 20 minutes at room temperature.

Application: Immunohistochemistry (IHC-P)

Species: Human
Tissue: Cervix well-differentiated squamous cell carcinoma
Sample: Paraffin-embedded section

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

Wash buffer: 1× TBST
Endogenous peroxidase blocking: 3% H₂O₂, 10 minutes at room temperature.
Blocking: 1% BSA + 10% normal goat serum, 10 minutes at room temperature.
Primary antibody: HA601131, 1/200, 1 hour at room temperature.
Secondary antibody: HA1120, 20 minutes at room temperature.

Application: Immunohistochemistry (IHC-P)

Species: Human
Tissue: High-grade serous ovarian carcinoma
Sample: Paraffin-embedded section

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

Wash buffer: 1× TBST
Endogenous peroxidase blocking: 3% H₂O₂, 10 minutes at room temperature.
Blocking: 1% BSA + 10% normal goat serum, 10 minutes at room temperature.
Primary antibody: HA601131, 1/500, 1 hour at room temperature.
Secondary antibody: HA1120, 20 minutes at room temperature.

Western blot analysis of p16INK4a on different lysates with Mouse anti-p16INK4a antibody (HA601131) at 1/1,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: HepG2 cell lysate
Lane 3: HEK-293 cell lysate
Lane 4: NIH:OVCAR-3 cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 16 kDa
Observed band size: 16 kDa

Exposure time: 2 minutes;

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 (HA601131) at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1:100,000 dilution was used for 1 hour at room temperature.

Immunocytochemistry analysis of HeLa cells labeling p16INK4a with Mouse anti-p16INK4a antibody (HA601131) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS 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 Mouse anti-p16INK4a antibody (HA601131) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Mouse IgG H&L (iFluor™ 488, HA1125) 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 (ET1602-4, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Rabbit IgG H&L (iFluor™ 594, HA1122) were used as the secondary antibody at 1/1,000 dilution.

Immunocytochemistry analysis of HeLa cells labeling p16INK4a with Mouse anti-p16INK4a antibody (HA601131) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS 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 Mouse anti-p16INK4a antibody (HA601131) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) 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.

Ki67 (HA721115, green) was stained at 1/250 dilution overnight at +4℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution.

Application: Immunohistochemistry (IHC-P)

Species: Human
Tissue: High-grade serous ovarian carcinoma
Sample: Paraffin-embedded section

Primary antibody dilution: 1/200

Antigen retrieval: ER2
Platform: Leica Biosystems BOND® RX

Application: Immunohistochemistry (IHC-P)

Species: Human
Tissue: Cervix well-differentiated squamous cell carcinoma
Sample: Paraffin-embedded section

Primary antibody dilution: 1/200

Antigen retrieval: ER2
Platform: Leica Biosystems BOND® RX

Application: Immunohistochemistry (IHC-P)

Species: Human
Tissue: Ovary Advanced serous carcinoma
Sample: Paraffin-embedded section

Primary antibody dilution: 1/200

Antigen retrieval: HIER (CC1)
Platform: Ventana® BenchMark ULTRA

Application: Immunofluorescence (IF-tissue)

Species: Human
Tissue: Tonsil
Sample: Paraffin-embedded section

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

Wash buffer: 1× PBS
Endogenous peroxidase blocking: 3% H₂O₂, 10 minutes.
Blocking: 1% BSA + 10% normal goat serum, 10 minutes at room temperature.
Primary antibody: HA601131, 1/500, overnight at 4℃.
Secondary antibody: Goat Anti-Mouse IgG (iFluor™ 488, HA1125), 1.5 hours at room temperature.