Anti mouse igg2b

Spheroids were rinsed in PBS before fixation in 10% neutral-buffered formalin (NBF). Spheroids were embedded in HistoGel before paraffin embedding and sectioning. In vivo tumors were fixed in NBF before paraffin embedding and sectioning. Heat-induced antigen retrieval was performed using pH 6 sodium citrate buffer. Tissue was blocked using 5% BSA before incubation with the following antibodies: PERK #5683, GRP78 #3177, and PDI #3501 were from Cell Signaling Technologies (MA, USA); KDEL 10C3 was from Enzo (Exeter, UK); and reovirus μ1C 10F6 and reovirus σ3 4F2 were from the Developmental Studies Hybridoma Bank (IA, USA). Alexa Fluor 488, 546, and 647 conjugates of anti-rabbit IgG H+L, anti-mouse IgG H+L, anti-mouse IgG2a, and anti-mouse IgG2b were from Thermo Fisher (UK). Spheroid sections were imaged on a Zeiss 710 confocal (Jena, Germany) with quantification using Cell Profiler v.3. Exposure settings were based on the minimum possible, which still allowed robust spheroid identification for segmentation. Controls were compared during exposure setup to ensure similar intensities across experiments. Stained sections from in vivo tumor samples were imaged on a Perkin Elmer Vectra 3.0 with spectral unmixing of images performed using Perkin Elmer Inform software. Image quantification was performed using Cell Profiler v.3 as described in the Results.

rSmLEV1.3 was coated onto Immunlon 4HBX 96 well plates at a concentration of 6.25 μg/ml as before. Experimental mouse sera were used to probe rSmLEV1.3 at a dilution 1:100 in wash buffer (PBS containing 0.05% Tween-20) with 1% bovine serum albumin (BSA). HRP-conjugated anti-mouse IgG₁, anti-mouse IgG_2b (Invitrogen) and anti-mouse IgE (ThermoFisher) were diluted 1:1,000 in wash buffer with 1% BSA. As a negative control, the secondary antibody alone was included on every plate. Development was achieved with 2,2′-azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS) substrate solution (Sigma Aldrich); the reaction was stopped after 15 min with 1% sodium dodecyl sulphate (SDS) and the optical density (OD) read at 405 nm, using a Polarstar Omega Plate reader (BMG Labtech, Offenburg, Germany). Statistical analysis of unmatched groups (vaccinated vs controls) was conducted using a Student’s t-test post-hoc analysis (least significant difference). Longitudinal statistical analysis of the same individuals (i.e., pre-, and post-vaccination) was analysed using a Wilcoxon signed-rank test.