Blocking buffer: 5% NFDM/TBST
Primary Ab dilution: 1:1000
Primary Ab incubation: 2 hours at room temperature
Lysate: HeLa, A431, A549, HCT-116
Protein loading quantity: 20 μg
Exposure time: 3 S
MW: 46-62 kDa

Tissue: Human breast
Section type: Formalin fixed & Paraffin -embedded section
Retrieval method: High temperature and high pressure
Retrieval buffer: Tris/EDTA buffer, pH 9.0
Primary Ab dilution: 1:1000
Primary Ab incubation: 1 hour at room temperature
Counter stain: Hematoxylin
Description: The brown color represents the positive signal observed with PTM-5388

Tissue: Human colon
Section type: Formalin fixed & Paraffin -embedded section
Retrieval method: High temperature and high pressure
Retrieval buffer: Tris/EDTA buffer, pH 9.0
Primary Ab dilution: 1:1000
Primary incubation condition: 1 hour at room temperature
Counter stain: Hematoxylin
Description: The brown color represents the positive signal observed with PTM-5388

Tissue: Human breast cancer
Section type: Formalin fixed & Paraffin -embedded section
Retrieval method: High temperature and high pressure
Retrieval buffer: Tris/EDTA buffer, pH 9.0
Primary Ab dilution: 1:1000
Primary incubation condition: 1 hour at room temperature
Counter stain: Hematoxylin
Description: The brown color represents the positive signal observed with PTM-5388

Tissue: Human colon cancer
Section type: Formalin fixed & Paraffin -embedded section
Retrieval method: High temperature and high pressure
Retrieval buffer: Tris/EDTA buffer, pH 9.0
Primary Ab dilution: 1:1000
Primary incubation condition: 1 hour at room temperature
Counter stain: Hematoxylin
Description: The brown color represents the positive signal observed with PTM-5388

Tissue: Human cervical carcinoma
Section type: Formalin fixed & Paraffin -embedded section
Retrieval method: High temperature and high pressure
Retrieval buffer: Tris/EDTA buffer, pH 9.0
Primary Ab dilution: 1:1000
Primary incubation condition: 1 hour at room temperature
Counter stain: Hematoxylin
Description: The brown color represents the positive signal observed with PTM-5388

Tissue: Human lung adenocarcinoma
Section type: Formalin fixed & Paraffin -embedded section
Retrieval method: High temperature and high pressure
Retrieval buffer: Tris/EDTA buffer, pH 9.0
Primary Ab dilution: 1:1000
Primary incubation condition: 1 hour at room temperature
Counter stain: Hematoxylin
Description: The brown color represents the positive signal observed with PTM-5388

Tissue: Human lung squamous carcinoma
Section type: Formalin fixed & Paraffin -embedded section
Retrieval method: High temperature and high pressure
Retrieval buffer: Tris/EDTA buffer, pH 9.0
Primary Ab dilution: 1:1000
Primary incubation condition: 1 hour at room temperature
Counter stain: Hematoxylin
Description: The brown color represents the positive signal observed with PTM-5388

Cell line: HeLa
Fixative: 100% Ice-cold methanol
Permeabilization: 0.1% TritonX-100
Primary Ab dilution: 1:50
Primary incubation condition: 4°C overnight
Nuclear counter stain: DAPI (Blue)
Description: The green color represents the positive signal observed with PTM-5388

Tissue: Human colon carcinoma
Section type: Formalin fixed & Paraffin-embedded section
Retrieval method: High temperature and high pressure
Retrieval buffer: Tris/EDTA buffer, pH 9.0. Heat mediated antigen retrieval (Tris/EDTAbuffer, pH 9.0, 20 minutes) was used in betweenrounds of tyramide signal amplification to remove the antibody from the previous round, toavoid any cross-reactivity.
Primary ab dilution: PTM-5388 (1:1000), PTM-5130 (1:500), PTM-5081 (1:1000)
Primary Ab incubation: 90 minutes at room temperature
Secondary ab: Anti-Rabbit and Mouse Polymer HRP (Ready to use)
Nuclear counter stain: DAPI (Blue)
Comment: Color red is the positive signal for Pan-Keratin ( PTM-5388, TSA 570), color greenis the positive signal for CD68 ( PTM-5130, TSA 520), color yellow is the positive signal for CD8 alpha ( PTM-5081, TSA 650). The sectionwas incubated in three rounds of staining; intheorder of PTM-5388,PTM-5130,PTM-5081; each using a separate fluorescent tyramidesignal amplification system.

Tissue: Human breast carcinoma
Section type: Formalin fixed & Paraffin-embedded section
Retrieval method: High temperature and high pressure
Retrieval buffer: Tris/EDTA buffer, pH 9.0. Heat mediated antigen retrieval (Tris/EDTA buffer, pH 9.0, 20 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity.
Primary ab dilution: PTM-5388 (1:1000), PTM-5130 (1:500), PTM-5081 (1:1000)
Primary Ab incubation: 90 minutes at room temperature
Secondary ab: Anti-Rabbit and Mouse Polymer HRP (Ready to use)
Nuclear counter stain: DAPI (Blue)
Comment: Color red is the positive signal for Pan-Keratin ( PTM-5388, TSA 570), color green is the positive signal for CD68( PTM-5130, TSA 520), color yellow is the positive signal for CD8 alpha ( PTM-5081, TSA 650). The section was incubated in three rounds of staining; in the order of PTM-5388,PTM-5130,PTM-5081; each
using a separate fluorescent tyramide signal amplification system.