Roti histol

For immunohistochemical detection of the H3.3-G34W mutation formalin fixed paraffin embedded GCT tissue sections were deparaffinized in Roti-Histol (Carl Roth GmbH, Karlsruhe, Germany) and rehydrated in isopropanol. Antigen retrieval was performed using Dako target retrieval solution pH 6 (Dako, Hamburg, Germany) for 5 min at 121 °C in a pressure cooker. Sections were blocked for 30 min at room temperature using PBS supplemented with 5% bovine serum albumin (BSA). The primary rabbit anti-H3G34W antibody (Active Motiv, Carlsbad, USA) was diluted 1:1000 in PBS/1% BSA and incubated over night at 4 °C. The signal was amplified using the BrightVision +Poly-AP kit (VWR, Darmstadt, Germany) according to the manufacturer´s instructions. Samples were counterstained with hematoxylin (Carl Roth GmbH) and mounted using Neo-Mount (Merck, Darmstadt, Germany). All used antibodies are listed in Supplementary Table 1.

Fluoro-Jade B (Millipore, Chemicon, USA) staining was performed according to the manufacturer’s indications with slight modifications. Sections were immersed in 100% ethanol for 5 minutes followed by 70% ethanol (2 minutes) and distilled water (2 minutes). The rehydration step was followed by incubation in 0.06% potassium permanganate (15 minutes), then rinsed in dH2O and transferred for 30 minutes in 0.0004% Fluoro-Jade B solution in the dark. Finally, the sections were rinsed three times in distilled water and cleared in Roti-Histol (Roth, Germany) before coverslipping with DPX (Fluka, Milwaukee, WI). On average, four region-matched sections were analyzed per brain.

Paraformaldehyde (PFA)  fixed brains (4% in 0.1 M PBS) were washed in 0.1 M PBS, dehydrated in ethanol solutions with increasing concentrations (70, 80, 90, 96, 100%, three times, 30 min each), cleared in xylol (three times, 15 min and overnight), incubated (twice at 60 °C, overnight and 2 h), and finally embedded in paraffin. Sagittal brain slices (8 μm) were mounted on poly-l-lysine-coated glass slides. Paraffin was removed by incubation of the slices with RotI-histol (2 × 10 min, Carl Roth GMBH) and subsequent treatment with ethanol 100, 96, and 70% (3 min, each) and rinsing in H₂O. Staining was performed in 0.1% cresyl violet solution for 5–10 min followed by short rinsing in H₂O and two brief washes (in 96% ethanol). After dehydration in 100% ethanol (twice, 3 min each) slices were cleared in RotI-histol (twice, 3 min each) and mounted in a permanent mounting medium (Eukit, Sigma-Aldrich).

Formalin-fixed and paraffin-embedded tumor tissue was deparaffinized in Roti-Histol (Carl Roth) and rehydrated in isopropanol before sections were incubated in a pressure cooker at 121 °C for 5 min in a coplin jar containing Dako target retrieval buffer pH 6 (Dako, Hamburg, Germany). Sections were cooled down to room temperature, blocked for 15 min in PBS supplemented with 5% BSA and incubated overnight at 4 °C with a rabbit anti-H3.3 G34W antibody (RevMab Biosciences, San Francisco, CA, USA) diluted 1:200 in PBS supplemented with 1% BSA. Signals were detected using a BrightVision plus kit (VWR International, Bruchsal, Germany) according to the manufacturer’s instructions. For visualization, the red substrate ImmPACT Vector Red (Linaris, Dossenheim, Germany) was used. After 30 min of staining, the samples were counterstained with methyl green, mounted with NeoMount (Merck-Millipore, Darmstadt, Germany), and photographed using a Keyence BZ-X800 microscope (Keyence, Neu-Isenburg, Germany).

To visualize deposited matrix, Masson’s trichrome staining was performed (Sigma Aldrich, #HT15). In brief, samples were fixed for 10 min in ice-cold acetone at −20 °C, and subsequently washed in dH₂O for 5 min. Then, samples were incubated overnight in Bouin’s solution (Sigma Aldrich, #HT10132) at the room temperature, and washed the next day under running tap water for 5 min. Samples were then immersed in Weigert’s iron hematoxylin solution (Sigma Aldrich, #HT1079) for 3 min, and again washed under running tap water for 5 min. Samples were incubated with Briebrich Scarlet-Acid Fuchsin solution for 5 min, rinsed in dH₂O, and incubated with Phosphotungstic/Phosphomolybdic acid solution for 5 min. Finally, samples were immersed in Aniline Blue solution for 10 min, washed in 1% acetic for 2 min, and further washed with dH₂O, and then dehydrated through an ethanol gradient. Samples were then dipped 8–10 times and cleared in Roti®-Histol (Carl Roth, #6640) and mounted with Roti®-Histokitt (Carl Roth, #6638).

Pancreatic sections (2 µm) were de-paraffinized and rehydrated in Roti-Histol (Carl Roth, Karlsruhe, Germany) and a decreasing serial solution of ethanol. Slides were heated in citrate buffer (10 mM citrate acid, 0.05% Tween 20) in a pressure cooker using a microwave for 30 min at 900 W followed by a cooling step (30 min, RT). Sections were incubated with blocking solution (5% BSA/TBST, 1 h, RT), followed by primary antibodies (1 h, RT). Rabbit anti-insulin (Abcam, Cambridge, UK, ab181547) was used as primary antibody. Hydrogen peroxide blocking was performed with a 0.3% solution (Carl Roth) for 15 min at RT, followed by incubation with HRP-labeled Goat Anti-Rabbit IgG (1 h, RT) (Abcam, ab205718). Before mounting with VectaMount Mounting Medium (H-5000), tissue sections were incubated with 3,3’-diaminobenzidine in chromogen solution (ImPACT DAB Peroxidase Substrate Kit, Vector Laboratories, Burlingame, CA, USA) and counterstained with hematoxylin (Carl Roth). Stained insulin in whole pancreatic sections were imaged with a MIRAX-MIDI Scanner (Carl Zeiss AG, Oberkochen, Germany), equally edited with Pannoramic Viewer and analyzed with ImageJ Software.

For histological analysis, fixed embryos were rinsed in PBS and dehydrated using ethanol. They were embedded in paraffin and sectioned transversally at 7 µm thickness.
Paraffin sections were deparaffinized with RotiHistol (Carl Roth, Karlsruhe, Germany) and rehydrated for staining.
For hematoxlin-eosin (HE) staining the sections were stained with hematoxylin for 15 min and eosin for 2 min (Carl Roth, Karlsruhe, Germany).
Masson-Goldner-Trichrome-Staining was used in order to differentiate the connective tissue. First, the nuclei were stained for 5 min using hematoxylin according to Weigert. Then, the trichromatic stain was performed using ponceau-acid fuchsin, phosphotungstic acid-orange G, and 0.2% light green (Carl Roth, Karlsruhe, Germany). For differentiation, 1% acetic acid was used.
After both staining procedures, the sections were dehydrated again and covered. The sections were analyzed microscopically and photographed using the virtual slide microscope VS120 (Olympus, Tokyo, Japan). Histological measurements and cell density calculation were performed with the OlyVia software (Version 2.9, Olympus, Tokyo, Japan) and QuPath (Open source software, Version 0.3.2). The beads were indicated by circles in order to improve visualization.