GSM7821251: P21_LIN28A.negative_Control 1.3; Mus musculus; OTHER
Sections from each tissue were generated at 5 μm thickness and prepared according to NanoString GeoMx NGS FFPE protocol with minor modifications. Briefly, slides were incubated at 60°C for 30-60 minutes. Then, slides were sequentially incubated for 5 mins in xylene three times, 100% EtOH two times, 95% EtOH one time, and 1X PBS one time. To retrieve the target antigen, hydrated slides were first placed in 95°C DEPC H2O for 10 seconds, followed by 99-100°C 1X Tris-EDTA (pH 9.0) for 20 mins. Slides then washed in 1X PBS for 5 mins and incubated in 0.1ug/mL proteinase K for 15 mins at 37°C. Slides were then washed for 5 mins in 1X PBS one time, 10% neutral buffered formalin (NBF) one time, NBF stop buffer (0.1M Tris EDTA, 0.01M Glycine) two times, and another 1X PBS once. For in situ hybridization, 200μL of Buffer R, 25μL of WTA probe mix, and 25μL of DEPC H2O were added per slide. The slides were covered with a glass coverslip and incubated overnight in a humidity chamber at 37°C. The next day, slides were briefly submerged in 2X saline-sodium citrate buffer (SSCB). Then, off-target probes were removed using stringent wash solution (equal part formamide and 4X SSCB) twice at 37°C for 25 mins each, followed by 2X SSCB two times for 2 mins each. Blocking was done by adding 200uL of Buffer W per slide and incibated inside a humidity chamber for 30 mins. Buffer W was then removed from the slide and replaced with unconjugated primary antibodies and SYTO13 mixture that was diluted to the appropriate concentration using Buffer W and incubated in a humidity chamber for 1 hr at RT. Slides were then quickly washed with 2x SSCB twice, followed by 3 mins 2x SSCB washes three times. Slides were then incubated with secondary antibodies diluted in Buffer W for 30 min at RT in a humidified chamber. Slides were then washed 2x in SSCB buffer and directly imaged for RNA collection using a NanoString GeoMx Digital Spatial Profiler (DSP) and GeoMx DSP Control center software (v. 3.0.0.109). For overall gene expression signatures between control and cKO, three circular regions of interest (ROIs) of approximately 600-650 mm in diameter were randomly placed over three different cross-sections of each animal for technical replication. For phenotype-specific regions, tubules were grouped based on containing only a single layer of spermatogonia or containing an incomplete compliment of spermatocytes within the tubule cross-sections. ROIs were then segmented to capture undifferentiated spermatogonia or advanced germ cells separately by thresholding fluorescent signal corresponding to SYTO13+LIN28A+DDX4+ and SYTO13+LIN28A-DDX4+ within each ROI. A minimum of 50 nuclei were captured within each segmented population. cDNA libraries were generated according to the manufacturers protocol. NanoString WTA