G9023

Sorted germ cells and SPZ_EJ were processed as described previously^{7 (link),38 (link)} and attached to UltraStick/UltraFrost Adhesion slides (Electron Microscopy Sciences). Samples were fixed in 4% PFA in PBS for 15 min before antigen retrieval in three consecutive 5-min incubations with boiling citrate (10 mM at pH 6), followed by triton X-100 (0.2% in PBS) for 15 min. After blocking for one hour in PBST with 5% normal goat serum (Merck G9023) and 0.1% BSA, antibodies were applied overnight at 4 °C in a humidified chamber. The primary antibodies were α-tubulin (Merck T9026; 1:1000), H3K9ac (Abcam ab4441; 1:1000), and Spo11 (Santa Cruz Biotechnology sc-33146; 1:1000). Anti-mouse or anti-rabbit IgG coupled with Alexa-555 (Invitrogen A-21422 and Merck AP510C, respectively) were applied for 1 h at room temperature and cells were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Merck G8294; 1:3000) before mounting with Fluoromount™.

Cells prepared on cover glasses were fixed with 4% PFA (Santa Cruz Biotechnology, Dallas, TX, USA) for 20 min and blocked in 10% normal goat serum (G9023; Merck) in PBSa (lacking MgCl₂ and CaCl₂). A primary mAb recognizing αvβ6 (clone 10D5, ab77906; Abcam Ltd.) and a secondary goat anti-mouse IgG antibody conjugated with Alexa Fluor 488 (Thermo Fisher) were used diluted in blocking buffer at dilutions of 1 in 200 and 1 in 500, respectively. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole; Sigma).

In total, 30 min after NTG exposure (vehicle 5% dextrose and 0.105% propylene glycol), the media was removed from the cells and the culture was washed with 1X PBS (D8662, Merck) 3 times^{79 (link)}. Afterwards, cells were fixed with 4% paraformaldehyde (158127, Merck) for 20 min at room temperature. Permeabilization was achieved with 0.1% v/v Triton 100X (P8787, Merck) for 5 min and blocking with 5% Normal Goat Serum (NGS, G9023, Merck) for 1 h, both in 1X PBS. Neurons were labeled with primary antibodies rabbit anti-MAP 1:250 (17490-1-AP, LabClinics, Barcelona, Spain) and mouse anti-CGRP 1:200 (AB81887, Abcam, Cambridge, UK) incubated overnight at 4 °C. Secondary antibodies goat anti-rabbit Alexa 488 1:1000 (A11034, Thermo Fisher Scientific) and goat anti-mouse Alexa 568 1:1000 (A11031, Thermo Fisher Scientific) were incubated for 1 h at room temperature and protected from light. Nuclei were stained with DAPI 1.5:10000 (D9564, Merck) for 5 min at room temperature. Slides where mounted with mowiol (475904, Merck) and images taken with a confocal microscope (LSM 900, ZEISS, Jena, Germany). Mean fluorescence intensity for each cell was obtained with ImageJ (Wayne Rasband, NIH), and the average value of positive cells was calculated for each picture.

Brain sections (10 µm) were cut with a Cryostat (Leica, Ashbourne, Co Meath, Ireland) and immediately transferred onto gelatin-coated slides. The brain sections were blocked in 3% donkey serum blocking solution (G9023; Sigma-Aldrich, Dublin, Ireland) prepared in PBS supplemented with 0.1% Triton-X (X100; Sigma-Aldrich, Dublin, Ireland) for two hours at room temperature, followed by overnight incubation at 4 °C with antibodies against GFAP (1:100, Z0334; Dako-Agilent Technologies Ireland Limited, Little Island, Ireland) and IBA1 (1:500, ab5076; Abcam, Amsterdam, the Netherlands). The following day, the sections were washed in PBS (3 times for 10 min) followed by incubation with secondary antibodies conjugated to donkey anti-rabbit IgG (AlexaFluor 488, Life tech, A2120, Thermo Fisher Scientific, Dublin, Ireland) or donkey anti-goat IgG (AlexaFluor 594, Life tech, A11058, Thermo Fisher Scientific Dublin, Ireland) for 2 h at room temperature. Sections were then washed and counter-stained with bizbenzimide (1:3000, B2261; Sigma-Aldrich, Dublin, Ireland).

Hornet brains were dissected out in PBS and fixed for 24 h at 4°C in 4% paraformaldehyde. The brains were washed 3 times (10 min each) in PBS solution containing 0.2% of Triton X-100 (PBST) and preincubated for 3 h at room temperature in PBST with 10% normal goat serum (G9023, Sigma-Aldrich, Steinheim, Germany), henceforth NGS/PBST, to avoid unspecific staining. Tissues were probed with rabbit anti-serotonin primary antibody (S5545, Sigma-Aldrich, Steinheim, Germany) diluted (1:250) in NGS/PBST for 7 days at 4°C. Then, the brains were washed 3 times (10 min each) in PBST and incubated in Alexa-fluor 488-conjugated goat anti-rabbit secondary antibody (A-11008, life technologies; diluted 1:200 in NGS/PBST) for 7 days at 4°C. According to manufacturer data, pre-incubation of the primary antibody with 500 μM serotonin inhibits specific staining.

Cytospin-slides were fixed in 100% acetone at room temperature for 15 min. For the collagen-1 staining, slides were pre-incubated with 10% normal goat serum (Sigma, G9023) in block buffer (1% Blocking Reagent, Roche, in PBS according to the manufacturer’s protocol) for 30 min. After rinsing with PBS, slides were incubated for 60 min with mouse anti-human collagen-1 (Abcam, ab6308, 1:2000) or Isotype control in block buffer. As second (goat anti-mouse antibody biotin-labeled) and third antibody (streptavidin, alkaline phosphatase (AP) conjugate) we used the Link-Label kit from Biogenex (link: HK-325-UM, label HK321-UK) according to the manufacturer’s protocol. To detect the collagen-1 positive cells we used New Fuchsin Alkaline Phosphatase Substrate Solution (0.01% New Fuchsin, 0.02% Sodium Nitrite, 0.03% Naphthol AS-BI Phosphate, 1 mM Levamisole, in 0.2 M Tris-HCl, pH 8.5). Cells were counterstained with hematoxylin (Sigma, Gill No. 3), dried and mounted in Vecta Mount (Vector, Burlingame, CA, USA). For CD15 detection we used the same protocol, but with different antibodies; slides were pre-incubated with normal rabbit serum (Sigma, R9133) and subsequently stained with mouse anti-human CD15-FITC (BD Biosciences, HI98) or isotype control and rat anti-FITC AP conjugate (Sigma, A4843).