Apc 028

Deeply anesthetized animals were perfused, and brains were postfixed in 4% PFA overnight followed by sequential immersion in 15% and 30% sucrose for cryoprotection. Brains were then sectioned (20 μm) and stained as previously described [27 (link)]. Briefly, sections were blocked in 0.1 M PBS containing 5% goat serum and 0.3% Triton X-100, and then incubated in primary antibody mixture including rabbit anti-SK2 (1 : 200; APC-028, Alomone Labs) and mouse anti-PSD95 (1 : 200; MA1-045, Invitrogen) in 0.1 M PBS containing 1% BSA and 0.3% Triton X-100 overnight at 4°C. Sections were washed 3 times (10 min each) in PBS and incubated in appropriate Alexa Fluor–conjugated secondary antibodies (Invitrogen) for 2 h at room temperature. Images were acquired using a Zeiss LSM880 AiryScan confocal microscope.

A large panel of anti-SK2 antibodies were raised in our laboratory. These antibodies together with commercially available antibodies from Abcam (ab85401 and ab48817), Thermo Fisher Scientific Pierce (PA5-19205) and Alomone (APC-028) were screened in immunoblot and immunohistological analysis.
Only two individual antibodies turned out to be suitable for human tissues. The N-terminal SK2 antibody used in our study (Anti-NSK2) was produced in our laboratory against the amino acid sequence 19–37 (ASRRNLHEMDSEAQPLQPP). This sequence is highly conserved within different species and, therefore, could be used for experiments with human and mouse tissue. Mouse and rat SK2 channels possess a homologous amino acid sequence in this region, while the human SK2 sequence differs only in a single amino acid at position 19. The antibody directed against the carboxy-terminus of the SK2 channel (Anti-CSK2) recognizes the amino acid sequence 421–469 of human SK2 channel (Anti-KCNN2, ab85401 from Abcam) (Chakroborty et al. 2012 (link)). Mouse and human SK2 channels have homologous amino acid sequences in this region.