Alexa fluor 488 affinipure donkey anti mouse igg

To confirm localization of neuronal nuclei (NeuN) and myelin basic protein (MBP) in neurons, the mouse brain sections were processed using double immunofluorescence staining. Tissue sections from 1.82 and 2.32 mm posterior to the bregma according to a mouse atlas (Paxinos & Franklin, 2001), separated by intervals of 150 μm, were obtained from each animal. Each tissue section was sequentially treated with 10% normal goat serum in 0.1 M PBS for 30 min at 25°C. The sections were incubated overnight with a mixture of antisera of mouse anti‐NeuN (1:100; Merck Millipore) and rabbit anti‐MBP (1:200; Merck Millipore) at 25°C. The next day, after washing in PBS three times, the sections were treated with a mixture of both Cy3‐conjugated donkey anti‐rabbit IgG (1:500; Jackson ImmunoResearch) and Alexa Fluor 488 AffiniPure Donkey Anti‐Mouse IgG (1:500; Jackson ImmunoResearch) for 2 hr at 25°C. Thereafter, the sections were mounted on gelatin‐coated slides and in a water‐soluble mounting medium, Fluoromount‐G^® (SouthernBiotech).

Flow cytometric analysis was performed as per the MISEV2018 guidelines outlined by the International Society of Extracellular Vesicles 11 (link). For analysis of single EVs using flow cytometry, EVs were stained with streptavidin-Alexa Fluor® 488 (Abcam) or Alexa Fluor® 488 AffiniPure donkey anti-mouse IgG (Jackson ImmunoResearch, USA). For assessing RNA loading, FAM-conjugated ASOs were loaded onto EVs and the FAM signal acquired using the 488 nm laser. EVs were washed twice in PBS, diluted 10,000-fold and analyzed using a NanoFCM system (NanoFCM, United Kingdom). The laser power was fixed at 8 mW and the SS decay at 10%. The sampling pressure was fixed at 1.0 kPa prior to acquisition and events were recorded for a duration of 1 minute for each sample. In addition to EV + antibody controls, reagent controls containing staining antibodies diluted accordingly in filtered PBS were compared to PBS only, to eliminate any contribution by aggregated antibodies/proteins. EV samples were lysed with Triton X-100 and reanalyzed after each run to ensure that events within the EV gate were actually caused by the presence of EVs and not by non-EV particles/aggregates.

Ovaries were dissected and ovarioles fixed as described above (see DAPI staining). Following fixation, ovarioles were blocked in 200 µl blocking solution for 2 h at room temperature. Ovarioles were transferred into 100 µl of PBSTx (PBS + 0.1% Triton X-100) containing mouse anti-Lamin primary antibody (1/100; ADL67.10; Developmental Studies Hybridoma Bank) and left rotating at room temperature overnight. The following day, ovarioles were washed three times in PBSTx for 10 min each and transferred into 100 µl secondary antibody solution (1/250 in PBSTx; Alexa Fluor 488 AffiniPure Donkey anti-mouse IgG; Jackson Immuno Research) including DAPI (0.4 µg/ml; Sigma). Incubation was allowed at room temperature overnight as before. The following day, ovarioles were washed (3 × 10 min in PBSTx) and mounted onto slides.

Paraffin embedded lung was sectioned and mounted on ProbeOn Plus slides (Thermo Fisher Scientific, Pittsburgh, PA). Sections were deparaffinized by heating in a 60 °C oven for 20 min and then immersing in three sequential xylene baths and rehydrated in descending alcohol solutions followed by distilled water. Antigen retrieval was achieved by placing slides in 1 mM ethylenediamine tetraacetic acid (EDTA), pH 8.0 and brought to boil via microwave. Non-specific staining was blocked with 10% donkey serum (Jackson Immuno Research Laboratories 017–000-121, West Grove, PA) in PBS. The primary antibodies, a 1:100 dilution of podoplanin (Novus Biologicals NB600-1015, Littleton, CO) in PBS and a 1:50 dilution of E-cadherin (BD Biosciences BD 610,181, San Jose, CA) in PBS, were applied via capillary action and incubated with the slides overnight at 4 °C. Non-specific binding was blocked with 10% donkey serum in PBS. DyLight 594 AffiniPure goat anti-syrian hamster IgG (Jackson Immuno Research Laboratories 107-515-142, West Grove, PA) and Alexa Fluor 488 AffiniPure donkey anti-mouse IgG (Jackson Immuno Research Laboratories 715-545-150, West Grove, PA) were the secondary antibodies.

Microglia and oligodendrocytes were washed three times with PBS, fixed with 4% paraformaldehyde for 10 min (oligodendrocytes) or 20 min (microglia), washed, permeabilized with 0.05–0.1% Triton X-100 for 10 min, rinsed, and blocked for 30 min in PBS/1% BSA. Microglia were stained for about 3 h at 37°C in 1% PBS/BSA with 5 μg/mL Cy2-phalloidin (Sigma-Aldrich), in combination with primary antibodies against P2Y₁₂ receptor, as reported in Table 1. Oligodendrocytes were stained with primary antibodies against P2Y₁₂ receptor and MBP or NG2. The secondary antibodies used for double labelling are Cy3-conjugated donkey anti-rabbit IgG (1 : 100, Jackson Immunoresearch) or Alexa Fluor 488-AffiniPure donkey anti-mouse IgG (1 : 200, Jackson Immunoresearch). Cells were extensively washed and stained with the nucleic acid blue dye, Hoechst 33342 (1 : 1000). After rinsing, cells were covered with Fluoromount medium (Sigma-Aldrich) and a coverslip and analyzed by confocal microscopy.