Ab181701

Using an immunogold immunohistochemistry protocol tissues were prepared for TEM [38 (link),39 (link),40 (link)]. The fixation of the samples was carried out with 4% paraformaldehyde in PBS, after which samples were washed in PBS. Samples were cut with a vibratome (Vibratome Series 1000, Pelco 101, Ted Pella, Inc., Redding, CA, USA) into 20 µm thick sections. After washing in PBS, sections were incubated first in 50% ethanol for permeabilization and then in primary antibody for 48 h at +4 °C: rabbit anti-Cx37/GJA4 (1:100, ab181701, Abcam, Cambridge, UK); rabbit anti-Cx40/GJA5 (1:100, ab213688, Abcam, Cambridge, UK); and goat anti-Cx43/GJA1 (1:300, ab87645, Abcam, Cambridge, UK). Next, sections were rinsed in PBS after which overnight incubation followed with gold-conjugated donkey anti-rabbit or anti-goat secondary antibody (1:1000, 711-205-152 and 705-185-147, both from Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). The size of the gold particles used was 12 nm for anti-rabbit and 4 nm for anti-goat antibodies. On the next day, sections were rinsed in PBS, post-fixed in 1% osmium tetroxide (1 h), and then dehydrated in ethanol and embedded in Durcupan ACM resin (Sigma-Aldrich Inc., St. Louis, Missouri, USA). The sections were observed with a transmission electron microscope (JEM JEOL 1400, Jeol Ltd., Tokyo, Japan).

After deparaffinization and rehydration, antigen retrieval was performed by incubating the sections of the
antral follicles in 0.01 M sodium citrate buffer (pH=6.0,
Sigma, USA) for 1 hour in a 96°C oven. The sections
were washed two times in PBS-T, and incubated in 10%
goat and donkey serums (Sigma, USA) diluted in PBS
for 1 hour at 37°C to block non-specific protein bindings
in Cx37 and Cx43 immunostainings, respectively. After
two PBS washes, the sections were incubated overnight
at 4°C with primary antibodies against Cx37 [Primary
rabbit polyclonal antibody (ab181701, Abcam, UK)]
and Cx43 [primary mouse monoclonal antibody (C8092,
Sigma, USA)]. Both primary antibodies were diluted
1:400 in related blocking solutions. Then, the sections
were rinsed with PBS carefully and incubated with secondary antibodies [(goat anti-rabbit IgG (H+L) crossadsorbed (Alexa Fluor 488, A11034, Thermo Fisher Scientific, USA) and donkey anti-mouse IgG H&L (Alexa
Fluor 488, ab150105, Abcam, UK)] for 1 hour at 37°C.
Both secondary antibodies were diluted 1:1000 in related blocking solutions. Lastly, the sections were washed,
counterstained with DAPI for 1 minute, and inspected
under an inverted fluorescence microscope (Eclipse 50i,
Nikon, Japan). Sections from rat heart tissue were used
as a positive control. For the negative control, ovarian
sections were processed without the primary antibodies.