Superfrost slide

Fresh anonymised lymphoid patch-containing human ileal tissue specimens used in this study were purchased from a commercial tissue bank (Tissue Solutions, UK) and held for use under HTA licence agreement 12383. Control samples were from the resection margins of patients with intestinal tumours (7 samples) or ulcerative colitis (2 samples), and Crohn’s disease samples (6 samples) were from patients with different disease anatomical locations (Supplementary Table 4). Following collection specimens were snap frozen and embedded in Optimal Cutting Temperature compound. Tissue sections were subsequently cryo-sectioned at 14 μm thickness, collected on SuperFrost® slides (Thermo Scientific, USA) and allowed to air dry for 1 hour at room temperature.

Immunostaining of the OE was performed on 12 μm coronal cryosections of paraformaldehyde-fixed tissue from adult C57BL6 mice on superfrost slides (Thermo Scientific, Menzel Gläser). After blocking with 1% fish gelatin in phosphate-buffered saline containing 0.1% Triton X-100, sections were incubated with primary antibody (anti-Paqr8 (Abcam): 1:100, anti-Pacr9 (Abcam): 1:50, anti-GPR30 (Abcam): 1:100, anti-Pgrmc1 (Sigma-Aldrich): 1:100) and fluorescently-labeled secondary antibody (Invitrogen, anti rabbit Alexa 456 nm, 1:100) dilutions in blocking solution. Stained sections were mounted in ProLong Antifade Gold medium (Molecular Probes). All fluorescence images were collected on a confocal laser scanning microscope (LSM510 Meta, Zeiss, Oberkochen, Germany). For the anti-Pgrmc1 antibody, the staining was performed on tissue sections following a heat-induced epitope antigen retrieval using citrate buffer (10 mM citric acid, 0.05% Tween 20, pH 6.0). Control experiments completed without addition of the primary antibody revealed a low level of background staining.

The tissue slides derive from archival formalin-fixed paraffin-embedded (FFPE) tissue samples from 95 female patients with ductal carcinoma in situ (DCIS) who have undergone minimally invasive vacuum-assisted percutaneous breast biopsy in Breast Unit, Lower Silesian Oncology Center (Wroclaw). The use of the material has been approved by the Director of Lower Silesian Oncology Center. Additionally archival hematoxylin–eosin slides for each case were retrieved to be assessed as a “second look” by the two board-certified pathologists experienced in breast cancer (AH and LF). The IHC reaction was conducted with rabbit monoclonal ready-to-use (2 µg/mL) antibody against human Ki-67 antigen (Clone 30-9) and ultraView Universal DAB Detection Kit (Ventana, Tucson, AZ, USA). The procedure was performed automatically using Benchmark XT (Ventana, Tucson, AZ, USA), in line with the producer’s manual. The 4-µm-thick paraffin sections were cut and mounted on SuperFrost Slides (Thermo Fisher Scientific, Gerhard Menzel GmbH, Braunschweig, Germany).
Research was conducted in accordance with local guidelines and regulations. The study was approved by local ethics committee (Bioethics Committee Wroclaw Medical University).

Procedures for tissue preparation, immunohistochemistry and in situ hybridization have been described in^{12 (link),65 (link),70 (link)}. Briefly, eyes were fixed in ice-cold 4% paraformaldehyde, rinsed with PBS, immersed in 30% sucrose overnight at 4 °C, embedded in Tissue Freezing Medium (EMS) and cryosectioned at 20 μm. For immunohistochemistry, antibodies were diluted in 3% donkey serum (Jackson, 017-000-121), and 0.3% Triton-X in PBS. Antibodies used for immunostaining were as follows: goat anti anti-CHX10 (1:300, Santa Cruz); rabbit anti-TFAP2A (1:500, DSHB); rabbit anti-Secretagogin (1:10,000; BioVendor). For in situ hybridization, sections were mounted on Superfrost slides (Thermo Scientific), treated with 1.5 mg/mL of proteinase K (NEB, P8107S), and then post-fixed and treated with acetic anhydride for deacetylation. Probe detection was performed with anti-DIG HRP (1:1000) and anti-DNP HRP (1:500), followed by tyramide amplification.

To investigate changes in the epidermal barrier function, we snap-froze the skin tissues on day 8 of the cultivation in Tissue-Tek O.C.T. compound (Sakura Finetek, Torrance, CA, USA) using liquid nitrogen and stored these at −80°C until use. Tissues were cut into 7-μm-thick sections at −20°C using a Microm HM 550 cryostat and mounted on Super-Frost slides (both from Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, cryosections were fixed for 10 min with ice-cold methanol (−20°C). Antibody (Ab) staining was performed as described by Hering et al. (52 (link)). The following antibodies were used: rabbit Ab against cytokeratin 10 (18343-1-AP) and involucrin (55328-1-AP; Proteintech Group, Rosemont, IL, USA) and mouse Ab against filaggrin (NBP2-53245-20; Novus Biologicals, Littleton, CO, USA) and E-cadherin (33-4000; Thermo Scientific, Waltham, MA, USA) at a concentration of 5 μg/ml. The appropriate secondary antibodies conjugated to Alexa Fluor 594 (red) (Molecular Probes, Eugene, OR) were applied (1:400 in phosphate-buffered saline with 0.1% Tween [PBST]) followed by Hoechst 33258 (Sigma-Aldrich, St. Louis, MO, USA) (1 μg/ml in Dulbecco's phosphate-buffered solution [DPBS]) application for counterstaining the nuclei. Sections were embedded in mounting medium and analyzed in biological triplicates using the LSM700 confocal microscope (Carl Zeiss, Oberkochen, Germany).

Formalin-Fixed, Paraffin-Embedded (FFPE) tongue and jejunum samples were sectioned onto Superfrost slides (Thermo Fisher Scientific, MA, USA), followed by deparaffinization and rehydrated according to standard protocols. Slides were then prepared for IHC with anti-Myeloperoxidase (MPO) (ab65871, Abcam, Australia) rabbit polyclonal antibody using mouse and rabbit specific HRP/DAB IHC detection kit—micro-polymer (ab236466, Abcam, Australia) according to manufacturer's instructions. Briefly, rehydrated slides were treated with hydrogen peroxide (Abcam, Australia), which was followed by heat-induced epitope-retrieval (HIER) with 0.1 M citrate buffer for 15 min. The sections were then incubated with the protein-blocking reagent (Abcam, Australia) for 10 min and then treated with a specific antibody for MPO (Abcam, Australia; 1:1250; 4 °C for overnight). Post PBS washing, the sections were incubated with goat anti-rabbit IgG secondary antibody (Abcam, Australia) at room temperature for 15 min and developed using HRP-conjugated DAB substrate (Abcam, UK), followed by counter-staining with hematoxylin. Grading was performed based on positive stain counting on the scanned digital images of the slides using QuPath open-source digital software v. 0.2.01^{125 (link)}.

The mice were euthanized via neck dislocation. The eyes were enucleated, the cornea of each eye was pierced with a needle, and then, the eye was immediately embedded in paraformaldehyde (4% PFA) for 2 h. They were then washed three times for 10 min with 0.1 M phosphate buffer saline (PBS). Ultimately, the eyes were cryoprotected using increased sucrose–PBS solutions (10–30%). They were placed in Tissue Tek (Sakura Europe, Barcelona, Spain), and retinal portions of 8 µm were taken with the help of a cryostat (Leica CM 1850 UV Ag protect, Leica Microsistemas S.L.U., Barcelona, Spain). The sections were placed on superfrost slides (Thermo Fisher Scientific, Braunschweig, Germany) and kept at −20 °C.