Cervix consists of two distinct KRT5+ stratified and KRT7+/8+ columnar epithelial lineages.

Panel D shown only.

Transition zone (TZ) including stratified and columnar epithelium from human (A,C) and mouse (B, D) cervix tissue sections immunolabeled with antibodies against KRT5 (ab193894) and KRT8; nuclei are shown in blue.

Organoids were washed five times with cold PBS to remove Matrigel before fixing with 4% paraformaldehyde for 1 h at room temperature (RT) followed by washing with PBS twice. Organoids were then subjected to dehydration in an ascending ethanol series followed by isopropanol and acetone for 20 min each. The dehydrated organoids were paraffin-embedded and 5 µM sections cut on a Microm HM 315 microtome. Mouse and human tissues were extensively washed with PBS and fixed using 4% PFA overnight at RT. Samples were subjected to dehydration in an ascending ethanol series followed by isopropanol and xylene (60 min each) followed by paraffinization using a Leica TP1020 tissue processor. The tissue was embedded and 5 µM sections cut on a microtome. For immunostaining, paraffin sections were deparaffinized and rehydrated, followed by treatment with antigen retrieval solution. Sections were blocked using blocking buffer (1% BSA and 2% FCS in PBS) for 1 h at RT. Primary antibodies were diluted in blocking buffer and incubated for 90 mins at RT followed by five PSB washes before 1 h incubation with secondary antibodies diluted in blocking buffer along with Hoechst or Draq5. Sections were washed with PBS five times and mounted. Images were acquired with a Leica TCS SP8 confocal microscope.

ab193894 staining Cytokeratin 5 in HACAT cells. The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 10% normal goat serum in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab193894 at 1/200 dilution (shown in green) and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 594), at 1/200 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Flow cytometry overlay histogram showing A431 cells stained with ab193894 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1 % PBS-Triton X-100 for 15 min. The cells were incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab193894) (1/500 dilution) for 30 min at 22°C.

Isotype control antibody (black line) was Rabbit IgG (monoclonal) Alexa Fluor 488® (ab199091) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.

This antibody gave a positive signal in A431 cells fixed with 4 % formaldehyde (10 min) / permeabilized with 0.1 % PBS-Triton X-100 for 15 min used under the same conditions.