Histogene staining solution

Mouse liver tissue was cryosectioned and mounted onto microscope slides. The number of laser pulses was varied from 1-5 and the diameter of the beam shaping iris was set to 2, 4, or 12 mm. For each combination tested, 20 positions on tissue were ablated. After laser ablation, the slides were removed from the stage, excess water was removed, and the tissues were stained with 100 μL of histogene staining solution (Applied Biosystems, Foster City, CA, USA) pipetted directly on top of the tissue and allowed to sit for approximately 2 minutes. The slide was then washed with 70% ethanol for 30 seconds followed by 100% ethanol for 30 seconds. Excess ethanol was removed and approximately 50 μL of Permount mounting medium (Fisher Scientific, Waltham, MA, USA) was pipetted onto the tissue and a coverslip was mounted on the slide. Digital images of the stained tissues with ablation spots were taken using an LMD7000 (Leica Microsystems, Buffalo Grove, IL, USA) with a 5× objective. The JPEG images were loaded into MATLAB 2016b (Mathworks, Natick, MA, USA) and circles were fitted to the ablation spots using the “imfindcircles” function in the Image Processing Toolbox.

The ZG layer was isolated from human adrenal glands using laser capture microdissection (Arcturus XT system, Life Technologies). Before laser capture microdissection, adrenal glands were incubated in RNAlater Solution (Life Technologies) at 4℃ overnight, embedded with OCT, sectioned into 20 μm slices, and stained with HistoGene Staining Solution (Life Technologies). RNA was extracted (RNeasy Micro Kit, QIAGEN), reverse transcribed (SMART-Seq v4 Ultra Low Input RNA Kit, Clontech Laboratories). PCR was performed in 20 μL reactions containing: 2 μL of template, 0.4 μmol/L of each paired primer, and iQ SYBR Green Supermix (Bio-Rad). The thermocycling conditions were 95℃, 3 min; 40 cycles of 95℃, 15 s; 58℃, 30 s; 72℃, 30 s; and 72℃, 10 min. Primers used in PCR: Ca_V1.1: forward 5’ CAGCAC TACAAC CAGTCG GA, reverse 5’ CTCCAC CCAGGC AATACA GTC; Ca_V1.2: forward 5’ CCTTTC TGGTTT AGCTGT GGG AAG ATCT, reverse 5’ AATGCA AAGAGT TACTGAT TCCCGT TTCAG; Ca_V1.3: forward 5’ GCACTA CGA GCA GTCCAA GA, reverse 5’ GCTGCC GATTAC GATGAG GG; Ca_V1.4: forward 5’ ACGGTG GAGATG CTTCTC AAATTG TACG, reverse 5’ GGTGAC CTTAAA GATCCT GAGGAG GC; Ca_V3.1: forward 5’ GATCCT GACCCA GGAGGA CT, reverse 5’ AAGAGC ACGTAG TTGCCGAA; Ca_V3.2: forward 5’ CCTGGA GGAGAG CAACAAGG, reverse 5’ GGTCTC CATCTC CACCTCCT; Ca_V3.3: forward 5’ ACGCAT CTTTCT CACCGTGT, reverse 5’ ATGGGC TTGAGG GAGGAGAT;