Goat serum blocking solution

Mouse ovarian sections were provided by BIOSERV Analytik (Rostock, Germany). Sections were deparaffinized and subjected to antigen retrieval using sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0). Sections were blocked with 10% goat serum blocking solution (Life Technologies, Frederick, MD, USA) at RT for 1 h, rinsed with PBS, and incubated in 1× mouse-on-mouse IgG blocking solution (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) at RT for 1 h. Sections were incubated with a 1:30 dilution of serum samples and kept at 4 °C overnight. The sections were washed and incubated with 10 µg/mL fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Invitrogen) as a secondary antibody for 1.5 h at 37 °C. After washing, the slides were mounted with DAPCO mounting medium (25 mg/mL DABCO, 90% glycerol, 10% PBS, pH 8.5) and observed under a fluorescence microscope. The sections were stained with methylene blue for bright-field microscopy.

To determine the immunoreactivities of the mitochondrial dynamics, biogenesis, synaptic, and autophagy/mitophagy proteins, immunofluorescence analysis was performed on brain sections from non-diabetic animals, diabetic animals that were fed with chaya, and control diabetic animals that were fed with regular chow. Details of the antibody dilutions are given in Table 3. Briefly, the frozen tissue sections were fixed in freshly prepared 4% paraformaldehyde in PBS for 15 min, and then washed with PBS and permeabilized with 0.1% Triton-X100 in PBS as described previously [17 (link),24 (link)]. They were blocked with 10% goat serum blocking solution (Life technologies) for 1 h at room temperature. All sections were incubated overnight with the antibodies and dilutions described in Table 3. After incubation, the sections were washed three times with PBS, for 10 min each time. The sections were incubated with a secondary antibody conjugated with Fluor 488 (Invitrogen) for 1 h at room temperature. The sections were then washed three times with PBS and mounted on slides. Photographs were taken with an Olympus IX 83 microscope. To quantify the immunoreactivities of antibodies, 10–15 photographs were taken at ×40 magnifications and statistical significance was assessed using cellSens software analysis.

Ovarian sections of immunized BALB/c and unimmunized wild mice were obtained from BIOSERV Analytik (Rostock, Germany). Ovarian sections were deparaffinized and subjected to antigen retrieval using sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0). Sections were blocked with 10% goat serum blocking solution (Life Technologies, Frederick, Maryland, USA) at RT for 1 h, washed with PBS, and incubated with 1X mouse-on-mouse IgG blocking solution (Invitrogen, Thermo Fisher Scientific) at RT for 1 h. Sections of unimmunized wild mice were incubated with a 1:20 dilution of serum samples from vaccinated BALB/c mice and kept at 4°C overnight. Sections of immunized BALB/c mice were incubated with a blocking solution (without the addition of any serum samples), washed, and incubated with 10 µg/mL fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG secondary antibody (Invitrogen, Thermo Fisher Scientific) at 37°C for 1.5 h. After washing, slides were mounted with the DABCO mounting medium (25 mg/mL DABCO, 90% glycerol, and 10% PBS; pH 8.5) and observed under a fluorescence microscope.