Observer z1 microscope

Bioprinted specimens were fixed in 3.7% formalin (Carl Roth) overnight at 4 °C and treated with a commercial tissue clearing kit (abcam, Cambridge, UK) according to the manufacturer’s instructions. Samples were analyzed for expression of cyclophilin B (abcam), pan-cytokeratin (Pan-CK; Santa Cruz Biotechnology, Inc., TX, USA), fibroblast specific protein 1 (FSP-1; LifeSpan BioSciences, Seattle, WA, USA) and pro-surfactant protein C (pro-SPC; Millipore, Billerica, MA, USA) Afterwards, the sample sections were incubated with corresponding Alexa 546- and Alexa 488-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA), and nuclear counterstaining was performed with 4′,6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific, Waltham, MA, USA). For imaging the samples were finally mounted in clearing buffer II (abcam) in a 3.5 mm silicon imaging chamber (abcam) and analyzed with the Observer Z1 microscope (Zeiss Observer Z1 microscope; Zeiss).

Normal ovarian and ovarian adenocarcinoma paraffin-embedded tissues were purchased from OriGene technologies (Maryland, USA). Paraffin was removed by soaking the slides in xylol before rehydrating the tissues with PBS for 20 min at room temperature. Next, 200–300 ml of pre-heated antigen retrieval buffer (1 mM EDTA pH=8) were added to the slides before heating twice for 5 min in the microwave (700 W). Following, the tissues were delimited using a clear nail polish and blocked using a 4% BSA blocking solution before incubation with the primary StarD13 antibody for 2 h. After washing the slides with ice-cold PBS1X, the tissues were incubated with the appropriate secondary antibody coupled to Alexa fluor-488 fluorophore for 30 min before mounted using a mounting solution mixed with DAPI. Fluorescent cell images were taken using the 20X objective lens of the fluorescent Zeiss Observer Z1 microscope operated by the Zen software (Oberkochen, Germany).

Fixation was carried out with ice-cold 4% paraformaldehyde (30 min), followed by permeabilization when necessary with 0.1% Triton X-100 on ice (10 min) and blocking in 3% bovine serum albumin (BSA;30 min). Incubation with primary antibodies was carried out in a 3% BSA solution (overnight) at 4 °C. Primary antibodies used were: rabbit anti-SIRT1(H300) (1:400; Santa Cruz, Cat. No. sc-15404), rabbit anti-NF-kB p65 (1:400; Thermofisher, Cat. No. PA1-186), mouse anti-HIF-1α(H1alpha-67) (1:80; Santa Cruz, Cat. No. sc-53546). After washing, cells were incubated with secondary antibodies for 45 min RT, washed, and mounted with 4′,6-diamidino-2-phenylindole (DAPI)-containing mounting solution (both from Sigma). Secondary antibodies used were: Alexa-Fluor 546-anti-mouse (1:300; Invitrogen), Alexa-Fluor 488-anti-rabbit (1:300; Invitrogen). Digital images were captured with a Zeiss Observer.Z1 microscope equipped with the Apotome.2 acquisition system (Zeiss, Oberkochen, Germany).

Bioprinted constructs were fixed in 3.7% formalin (Sigma), embedded in paraffin, and sliced into 10 μm thick sections. After deparaffinization, antigen retrieval was performed in Tris-EDTA (10 mM Tris Base, 1 mM EDTA, pH 9.0) at 95 °C for 30 min. Cells were permeabilized using 1% Triton-X-100 for 15 min, and blocking was performed for 30 min using 5% goat serum. For the characterization of nuclear damage, γH2AX (anti-γH2A.X (phosphor S139), Abcam, ab11174, 1:1000, Berlin, Germany) was used. For the cellular characterization of epithelial cells, pan-cytokeratin (anti-pan Cytokeratin, abcam, ab27988, 1:250) was used. This was followed by incubation with corresponding secondary antibodies (Alexa Fluor 546- or 488-conjugated anti-rabbit or anti-mouse IgG(H+L) (A11005, Thermo Fisher Scientific, Waltham, MA, USA; 1:2000). Nuclear counter-staining was performed with DAPI (Sigma), and slides were mounted in Mowiol 4–88 (Roth). The slides were analysed by fluorescence microscopy (Zeiss Observer, Z1 microscope, Carl Zeiss, Zeiss, Germany).

Cryosections of 20 μm were fixed in cold methanol for 10 min and blocked for 1 h with normal goat serum, followed by tissue permeabilization in 0.1% Tween 20 in PBS for 5 min. Then the sections were incubated overnight with rat anti-mouse CD31 (BD Biosciences, Catalog No. 553370; 1.25 μg/ml) or polyclonal rabbit anti-Aβ40 antibody (Agrisera; 0.5 μg/ml) at 4 °C, and incubated with Alexa-594-conjugated goat anti-mouse IgG or Alexa-488 goat anti-rat IgG for 1 h at room temperature.
NTE was performed in darkness according to the previously described protocol [19 (link)]. Following immunostaining, the sections were directly submerged in ILFORD K5 emulsion, air-dried for 2 h at room temperature and exposed for 2 weeks at 4 °C. The emulsion-covered tissue sections were developed according to the manufacturer’s instructions, dehydrated in an increased series of ethanol solution and mounted with Pertex mounting medium. The immunofluorescence and NTE stainings were imaged with a Zeiss Observer Z.1 microscope using the ZEN 2.6 software (Carl Zeiss Microimaging GmbH, Jena, Germany).

Nuclear track emulsion (NTE) allows visualisation of radiolabelled molecular compounds as silver grains on top of immuno-stained sections. NTE experiments were done in darkness, as previously described [21 (link)]. In brief, ILFORD K5 emulsion (Oxford Instruments, Gometz la Ville, France) was prepared in a 40 °C water bath as a 50:50 emulsion in MQ-water. The CD31-stained sections were immersed in the emulsion for 10 s. The sections were left to air dry for 2 h, before they were incubated in dark conditions for 5 weeks at 4 °C. The sections were developed according to the manufacturer’s instructions, then dehydrated in increasing EtOH concentration gradient (70%, 95%, 100%) and mounted with Pertex (Histolab). Images of the developed emulsion and CD31-immunofluorescent stained sections were acquired with a Zeiss Observer Z.1 microscope (Carl Zeiss Microimaging GmbH, Jena, Germany) and processed equally using the ZEN software. An alternative version of Fig. 7a, b, can be found in the supplementary material with colors converted by the function “Selective Color” in Photoshop, to show silver grains in white on a dark background (Additional file 1: Fig. S6).