In Situ Hybridization of Kifc1 in Mouse Testes

The ISH was conducted as described by Helmprobst et al. (2017) [48 (link)]. The testes were collected and fixed in 4% paraformaldehyde (PFA in PBS, pH 7.4) in the 4°C for 2 hours. After rinsing in PBS, the samples were dehydrated in 0.5 M sucrose (dissolved in PBS) in 4°C for another 2 hours, then embedded by Tissue-Tek O.C.T. Compound to store in the -40°C. For ISH, the samples were cut into 10 μm sections with Cryostat microtome. A length of 502 bp kifc1 fragment was cloned from cDNA and ligated to PGEM-T Vector with Sp6 and T7 transcription sites. Sense and antisense probe was transcribed with Sp6 and T7 RNA transcription enzymes (Promega, Madison, Wisconsin, USA) and the NTP DIG RNA labeling Mix (Roche, Basel, Switzerland), according to the Manufacturer's protocols. After hybridization with the DIG-RNA antisense probes, the slides were incubated with Anti-Digoxigenin-AP Fab fragments (Roche, Basel, Switzerland) for overnight, then stained with NBT/BCIP mix (Promega, Madison, Wisconsin, USA). The slides were then dehydrated in gradient alcohol as 50%, 70%, 90%, and 100% for 15 min each and permuted by xylene before sealed with neutral balsam. The sense probes were used as negative control. The pictures were taken by microscope (Olympus BX40). The bands were analyzed by software Image J.