Omniprep solution

Stainings were conducted as described previously by Harari-Steinberg et al. [58 (link)]. Paraffin blocks were cut into five µm sections. Sections were pre-treated using OmniPrep solution (pH 9.0) at 95 °C for one hour following the manufacturer protocol (Zytomed Systems, Berlin, 10587, Germany). Next, incubation with Cas-Block solution (Catalog number: 008120) (Thermo Fisher Scientific Waltham, MA, 02451, USA) for 20 min was used for blocking. Then, the slides were incubated O/N 4 °C with anti-RAP2B (Anti-RAP2B antibody number ab101369) diluted 1:500. Detection was done using ImmPRESS™ Anti-Rabbit Ig Reagent Peroxidase (VE-MP-7401) and developed with ImmPACT™ DAB peroxidase (Catalog number: SK-4100) (Vector Laboratories, Inc., Burlingame, CA, 94560, USA).

A total of 10⁶ cells were fixed in 4% paraformaldehyde (PFA) for 2 hr, washed in PBS, and suspended in low-melting-point agar. After becoming solid at 40°C the agar block was transferred into PFA overnight at 40°C, washed with distilled water, and embedded. Five-micrometer sections of paraffin-embedded agar blocks were mounted on super frost-plus glass and incubated at 60°C overnight. Following deparaffinization and antigen retrieval with Zytomed Systems OmniPrep Solution, slides were incubated in Cas-Block solution for 1 hr at room temperature. The primary antibodies SIX2 (11562-1-AP, Proteintech) and Ki67 (VP-K452, Vector Laboratories) were diluted in commercial antibody diluent, incubated for 1 hr at room temperature, washed with PBS-Tween (0.05%), and incubated with secondary antibodies for 1 hr at room temperature. Following PBS-Tween washes, slides were mounted with DAPI-containing mounting and covered with coverslips. Staining was evaluated with an Olympus BX51TF microscope and Scion color and monochrome digital cameras.
For experimental procedures for immunofluorescence staining of hFK cells, in vivo WT xenograft formation, single-cell gene expression analysis, and statistical analyses, see Supplemental Experimental Procedure.