Discovery xt

The IHC was performed using the streptavidin-peroxidase method with an automated immunostainer (DiscoveryXT; Roche, Penzberg, Germany) in paraffin-embedded tissue. After heat-induced antigen retrieval with EDTA (pH 8), we used the primary antibody anti-serotonin (5-HT) ABIN617893 from https://www.antibodies-online.com/antibody/617893/anti-5-Hydroxytryptamine in a dilution 1:100. We repeated the procedure two times with an appropriate positive control (neuroendocrine cells in murine gut) and a negative control (without the primary antibody) to confirm the specificity of the staining. Secondary antibody and blocking against nonspecific binding was performed according to antibody information. DAB and hematoxylin as contrast were used for visualization of the reaction peptide-hormone-antibody. Cytoplasmic staining was considered a positive reaction. Images for illustration were made by the slide-scanning system, NanoZoomer 2.0 HT (Hamamatsu Photonics K.K.; Hamamatsu City, Japan).

For immunohistochemistry on mouse organs, liver, heart and brain were collected from four Uqcrh^−/− and four wild‐type mice (two females and two males per group) at 9 weeks of age at the time of necropsy. The tissues were fixed in formalin for 24 h and embedded in paraffin for histological and immunohistochemical analysis. Immunohistochemistry was performed on 2 µm‐tick sections in an automated immunostainer (Discovery^®XT, Roche, Penzberg, Germany). Briefly, sections were incubated with primary antibodies against UQCRC2 (1:1,000; Abcam) and VDAC1/porin (1:2,000; Abcam) with a streptavidin–peroxidase detection reagent. As a negative control, the primary antibody was omitted. Digital images were captured with the NanoZoomer^® 2.0HT (Hamamatsu, Japan) digital slide scanner. A scoring system was used to quantify the expression levels of UQCRC2 and VDAC1/Porin: 0 = no staining; 1 = weak staining; 2 = moderate staining; 3 = strong staining. The score for each section was calculated as the mean of 4 high‐power fields (Vidali et al, 2017 (link)).

Tissue sections (5 µm) were cut onto charged slides and baked overnight at 65 °C. A protocol was designed on the Discovery XT machine (Roche Ventana, Arizona, USA) according to optimum antibody conditions (Table 2). Unique barcoded labels corresponding to the specific protocol were created and attached to the charged slides. The slides were inserted into the auto-stainer along with the required reagents. Upon protocol completion, the slides were removed and washed with Dako 1× wash buffer and distilled water to remove residual liquid coverslip. The slides were then placed in haematoxylin for 5–6 s and subsequently rinsed in running water to remove excess dye. The slides were placed in 70% EtOH for 1 min, 100% EtOH for 1–2 min, xylene for 2 min and cover-slipped for analysis.Table 2

IHC antibodies.

	Incubation time (min)	Optimal dilution	Company	Ref. code
1° antibody
Anti-ERG Rabbit mAB	32	Neat	Roche	EPR3864
Anti-Ki-67 Mouse mAB	32	1:80	Dako	IS62630-2
Anti-CCND1 Rabbit mAB	24	Neat	Dako	IS08330-2
Anti-Cytokeratin (Cam5.2) Rabbit mAB	16	Neat	Roche	790-4555
Anti-E-cadherin Mouse mAB	32	1:50	Invitrogen	18-0223
Anti-CD34 Mouse mAB	32	1:50	Dako	IR63261-2
Anti-Vimentin Mouse mAB	16	Neat	Dako	IR63061-2
2° antibody
OmniMap anti-Rabbit HRP	20	Neat	Roche	760-4311
Universal Secondary Antibody MultiLink	20	Neat	Dako	E045301-2

Four μm-thick sections of formalin-fixed paraffin-embedded (FFPE) explant tissue were stained for the proliferative marker Ki67. Automated immunohistochemistry (IHC) was carried out on a Discovery XT staining module (Roche). Antigen was retrieved by incubating slides in Cell Conditioning Solution (Roche). Endogenous peroxidase activity was reduced by incubating slides in Inhibitor CM (Roche). Slides were incubated in Ki67 primary antibody (Novocastra; 1:100) or Anit-AR primary antibody (clone G122-434, BD) for 1 h at 37°C followed by HRP-conjugated Discovery OmniMaP HRP secondary antibody (Roche) for 30 min. A ChromoMap DAB kit (Roche) was used for detection. Slides were counterstained in hematoxylin and mounted in DPX. Stained slides were imaged using an Aperio CS2 Digital Pathology scanner (Leica). Regions of interest (i.e. epithelial cells) were manually annotated and the percentage of Ki67 positive cells was determined using Aperio Image Analysis software (Leica) in a minimum of four representative 40× magnification regions containing a total of at least 1000 nuclei. Positive control tissue for Ki67 is shown in Supplementary Figure S8 alongside no primary control staining. Serial sections were also stained for p63 (basal cell marker identifying benign regions) and AMACR (cancer cell marker) to define regions of cancer (Supplementary Figure S9).

Immunohistochemistry (IHC) was performed using the DAB-Map-Kit (Roche) on 2–4 μm thick sections of formalin-fixed paraffin embedded PCLS after cell conditioning with EDTA antigen retrieval solution pH 8.4. Sections were loaded into the DiscoveryXT autostainer (Roche), deparaffinized, rehydrated and incubated with the anti-TMPRSS2 antibody [EPR3861] (Abcam) in a dilution of 1 in 1,000 for 32 minutes at 37°C followed by incubation with the secondary antibody (universal secondary antibody, Ventana, Roche) for 24 minutes. Diaminobenzidin (Ventana/View DAB, Detection Kit, Roche) was applied as chromagen according to the supplier´s instructions. Slides were counterstained with hematoxylin and bluing reagent before examination by light microscopy. Tissue sections from small intestine of healthy rhesus macaques served as positive control samples. Negative control staining was performed by omission of the primary antibody for immunohistochemistry.

Testicular and epididymal tissues were obtained from freshly slaughtered wild type Holstein cattle and they were immediately fixed in 4 % formaldehyde for 48 h. Immunohistochemistry (IHC) was performed on paraffin-embedded sections, including testicular parenchyma, as well as ductuli efferentes, epididymal head, corpus, and tail with efferent ducts and epididymal duct, respectively. The primary polyclonal antibody was directed against the PAC-ARK peptide and it was generated in the rabbit according to standard protocols (Davids Biotechnologie GmbH, Regensburg, Germany). IHC was performed in an automated immunostaining system (Discovery XT, Roche Diagnostics GmbH, Mannheim, Germany) at a dilution of 1:1000 while using the SABC (streptavidin-biotin-complex) method, mild EDTA (ethylenediaminetetraacetic acid) pretreatment, and DAB (diaminobenzidine tetrahydrochloride) for signal detection (DAB Map Kit, Roche Diagnostics GmbH, Mannheim, Germany). A rabbit IgG isotype control (ABIN3023746, antibodies-online GmbH, Aachen, Germany) was included at the same concentration as the primary antibody for confirmation of primary antibody specificity. Additionally, pure antibody diluent instead of primary antibody was applied to the control sections for an evaluation of non-specific binding of the secondary antibody.