Anti mouse igg alexa fluor 594 conjugate

An. stephensi, An. gambiae and Ae. albopictus eggs were fixed with 4 % paraformaldehyde for 10 min at 4 °C and washed twice with PBS 1X. The slides were incubated in 1 % Bovine Serum Albumine (BSA) in 1X PBS for 30 min at room temperature and successively for 1 h at 37 °C with anti-Asaia monoclonal antibody (patent pending N. MI2012A001529) diluted 1:1000. After three washings in PBS 1X, they were incubated for 30 min at 37 °C with anti-mouse IgG Alexa Fluor 594 conjugate (Invitrogen, Carlsbard, California, USA) diluted 1:100 in 1 % BSA in PBS and washed three times for 10 min with 1X PBS. Slides were mounted with glycerol and visualized by epifluorescent microscopy (Carl Zeiss Axio Observer.Z1, Milan, Italy).

Cerebral organoids were fixed in 4% paraformaldehyde (PFA) overnight at 4 °C, then washed in PBS for 10 min and dehydrated by incubations in Ethanol (70%, 95% then 100%) for 1 h at 4 °C followed by two times 1 h incubation with Xylene 100% at room temperature. The cerebral organoids were then embedded in Paraplast Plus (Leica, 39602004) and sectioned at 30 µm. Tissue sections were stained with Haematoxylin and Eosin (H&E) or used for immunostaining. For immunofluorescence, antigen retrieval was performed by using the Antigen Retriever buffer (Citrate Buffer pH 6.0, Sigma, C 9999, 10× ). Sections were then blocked and permeabilized in blocking buffer (0.5% Triton X-100 and 1% BSA in PBS) for 20 min. Sections were then incubated with primary antibodies in blocking buffer at the following dilutions: SOX2 (mouse, R&D systems, MAB2018, 1:200), TUJ1 (mouse, Biolegend MMS-435P, 1:3000), GFAP (Rabbit, Dako Z0334, 1/2500), PCNA (Rabbit, abcam ab18197, 1 µg mL⁻¹). For visualization, an antibody anti-mouse immunoglobulin G (anti–mouse IgG) Alexa Fluor 594 conjugate (Invitrogen, Molecular Probes) and an anti-rabbit IgG Alexa Fluor 488 conjugate (Invitrogen, Molecular Probes) was applied. DNA was stained by DAPI (1/500) and sections were mounted in ProLong Diamond Antifade mountant (Thermo Fisher Scientific, P36965). Images were collected by using an Olympus FV3000 RS with a 20x objective.

Anti-RTEL1, produced in Rabbit (Sigma, Cat. No. HPA067329); Anti-RPA1, produced in Mouse (Sigma, Cat. No. SAB1406399); Anti-RPA 32 kDa subunit (9H8), produced in Mouse (Santa Cruz Biotechnology, Cat. No. sc-56770); Anti-HA tag (C29F4), produced in Rabbit (Cell Signalling Technology, Cat. No. 3724); Anti-gamma H2A.X (phospho S139) antibody [3F2], produced in mouse (Abcam, Cat. No. ab22551); Anti-beta Actin, produced in Mouse (Abcam, Cat. No. ab8226); Anti-Rabbit IgG, HRP-conjugate (Millipore, Cat. No. 12–348); Anti-Mouse IgG, HRP-conjugate (Cell Signalling Technology, Cat. No. 7076); Anti-Rabbit IgG, Alexa Fluor™ 488-conjugate (Invitrogen, Cat. No. A-11008); Anti-Rabbit IgG, Alexa Fluor™ 594-conjugate (Invitrogen, Cat. No. SA5-10040); Anti-Mouse IgG, Alexa Fluor™ 488-conjugate (Invitrogen, Cat. No. A-11001); Anti-Mouse IgG, Alexa Fluor™ 594-conjugate (Invitrogen, Cat. No. A-11032)