Ultra 5 block

The tissue sections were prepared as earlier described. The tissue was encircled with a PAP pen and blocked for 1 h with Ultra V Block (TA-060-UB, Thermo scientific). Approximately 25 µg antibody fragments were dissolved in Ultra V Block, 10 % goat serum and 1:100 anti-CK19 to a total volume of 100 µL and added to the encircled area. Incubation was performed for 3 h in humid chambers. The liquid was removed by aspiration, and the slide was washed four times 1 min in PBS. The slide was incubated for 30 min in the dark with mouse Cy3-conjugated anti-c-Myc antibody [9E10] 1:250 (Sigma-Aldrich), Alexa Fluor 488-conjugated goat anti-mouse IgG2a 1:500 (Invitrogen), DAPI 1:1000 (Invitrogen) and 10 % goat serum dissolved in Ultra V Block to 100 µL. Alternatively, the antibody fragments above were replaced with an anti-Ki67 antibody (Abcam) in a dilution of 1:100, and as secondary antibody, a goat anti-rabbit antibody coupled to Alexa 546 (Life technologies) was added in dilution of 1:500. The slide was washed three times 1 min in PBS and mounted with Fluoromount mounting media (Sigma-Aldrich) and cover glass.

Uterine tissues fixed in 5% (v/v) formaldehyde (Merck, USA) for 24 h were dehydrated through a graded ethanol series and embedded in paraffin. Five micrometer cut uterine sections were taken. After deparaffinization and rehydration, citrate buffer (pH 6.0) was used for antigen retrieval using microwave. Subsequently, the slides were washed in PBS and endogenous peroxidase activity was blocked by 3% hydrogen peroxide in methanol for 15 mins at room temperature. After washing with PBS, the sections were treated with ultra V block (Thermo, UK; cat no:TA-125-UB,) and incubated with rabbit FoxO1 primary antibody (1:100 dilution; Cell Signaling, USA; cat no:2880S) overnight at 4°C. Next day, after washing out the primary antibody, slides were incubated for 60 min at room temperature with anti-rabbit secondary antibody (Vector, USA; BA-1000, 1/500 μL,) followed by incubation with horseradish peroxidase conjugated Streptavidin (Thermo Scientific, UK; TS-125-HR) for 30 min at room temperature. All incubation steps were performed in a humidified chamber to avoid dehydration of the slides. Positive immunoreactions were visualized with diaminobenzidine (DAB)-peroxidase substrate (Sigma; cat no:D4168) and counterstaining was performed with hematoxylin. FoxO1 expression was evaluated and photographed under a Zeiss (Oberkochen, Germany) light microscope.

For immunohistochemistry, formalin-fixed and paraffin-embedded (FFPE) sections were used. After deparaffinization, tissue sections were pretreated with citrate buffer (pH 6) for antigen retrieval and incubated with hydrogen peroxide block (TA-060-HP, ThermoFischer Scientific, Waltham, MA, USA) and Ultra V Block (ThermoFischer Scientific) to avoid unspecific reactions. Immunostaining was performed using a rabbit polyclonal anti-HSF1 antibody (Sigma-Aldrich Cat# HPA008888, RRID:AB_1079088; 1:500) in a moist chamber at room temperature for 30 min, following incubation overnight at 4 °C. Slides were washed between steps with Tris-buffered saline (TBS). Immunoreactions were visualized with the N-Histofine Simple Stain MAX PO System (#414142F, Nichirei Bioscience, Tokyo, Japan) and DAB substrate (#SK-4100, Vector Laboratories, Burlingame, CA, USA). The specimens were counterstained with hematoxylin (#MHS128, Merck, Darmstadt, Germany). The omission of the primary antibody served as a negative control. For evaluation, sections were assessed using the intensity of staining (S) (0: no staining; +1: weak; +2 moderate, +3 strong) and the average percentage of immunoreactive cells (P) was graded as 0 (negative), 1: <10%; 2: 10–50%, 3: 51–80%; 4: ≥81%. The expression score (ES) was calculated by the equation ES = P × S.

C/EBPδ stainings were performed essentially as described before [54 (link),55 (link),56 (link),57 (link)] with minor modifications. Four micron-thick paraffin embedded tissue sections were deparaffinized and treated with 0.3% H₂O₂ in methanol for 15 min to block endogenous peroxidase activity. Subsequently, slides were blocked with Ultra V block (#TA-125-UB; Thermo Fisher Scientific, Waltham, MA, USA) and incubated with a rabbit polyclonal antibody against C/EBPδ (#GWB-MM818H; GenWay Biotech, San Diego, CA, USA) in a 1:1000 dilution in PBS at 4 °C overnight. The next day, slides were incubated with Powervision poly-HRP anti rabbit IgG (#DPVM-55HRP; Immunologic, Duiven, Netherlands) for 30 min at room temperature and stained using 3,3’Diaminobenzidine (Bright DAB, #BS04-999; Immunologic). Hematoxylin (1:10 in demineralized H2O) was applied as counterstaining.

Cells were fixed in 4% paraformaldehyde for 15–20-min, permeabilized with 0.5% triton-X100, blocked with UltraV block (ThermoScientific) for 10-min and incubated with primary antibodies diluted in 0.1% Tween in PBS for 3 h at room temperature or overnight at 4°C. Following 30–60-min incubation with secondary antibodies at room temperature samples were mounted with Vectashield with DAPI (Vector Laboratories) and covered with glass coverslips. Antibodies are provided in Supplementary Table S5.