Apotome 2 function

In situ hybridization was performed as previously described [9 (link)]. Briefly, the eyes of one-month-old animals were enucleated and fixed for 4 h in 4% PFA. After washing in phosphate buffer (PB, 0.1 M, pH 7.4), the eyes were embedded in paraffin according to standard protocols. For in situ hybridization (ACD, Newark, NJ, USA), 6 mm thick paraffin sections were pre-treated with retrieval reagent and protease according to the user manual. BaseScope^TM Detection Reagent Kit v2—RED was used to label TGF-β receptor type 2 (Tgfbr2) (ACD catalog number: 845871). The sections were analyzed on an Axio Imager Z1 microscope with the Apotome.2 function (Carl Zeiss, Jena, Germany) using Zeiss Zen software (Carl Zeiss, Jena, Germany).

The techniques employed have been previously described^{19 (link)}. Briefly, 5-μm sections were incubated first with primary antibodies overnight at 4 °C, then for 1 h at room temperature (RT) with biotinylated secondary antibodies and finally with fluorochrome-labelled (BioLegend) or peroxidase-labelled streptavidin (Beckman Coulter). Peroxidase activity was revealed with 0.025% 3,3-diaminobenzidine (Sigma-Aldrich) in PBS containing 0.03% hydrogen peroxide. Low amounts of antigens (CD32 and ACE) were revealed by Tyramide signal amplification biotin or fluorescence amplification systems (Akoya, Biosciences). An isotype-matched negative control was performed for each immunostaining. When 3,3-diaminobenzidine was used on slides, they were counterstained with Gill’s haematoxylin (Sigma-Aldrich), mounted in XAM neutral medium (BDH Laboratory Supplies), analysed and imaged using an Optiphot 2 microscope (Nikon). Immunofluorescence-stained sections were cover-slipped in Prolong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific) and analysed with an Axio Imager M2 microscope coupled to a Hamamatsu’s camera Orca Flash 4v3 using the ApoTome.2 function (Zeiss) for optical sectioning. The antibodies employed are listed in Supplementary Table 19.