Immedge hydrophobic barrier pen

Pre‐prepared 10 μm human hair follicle tissue cryosections on SuperFrost Plus slides (Thermo Scientific, #10149870) were fixed in ice‐cold acetone for 10 min and allowed to dry at room temperature. ImmEdge Hydrophobic Barrier Pen (Vector labs #H‐4000) was used to draw a barrier around individual tissue sections and allowed to dry. Tissue sections were treated overnight with primary antibody diluted in phosphate‐buffered saline (PBS) within a humidified chamber kept at 4°C. Subsequently, excess solution was gently tapped off and tissue sections were washed twice with PBS. Sections were treated with fluorescent secondary antibody diluted 1:200 in PBS for 45 min at room temperature and protected from light. Slides were washed twice with PBS and stained with DAPI (1 μg/ml) for 2 min or, where indicated, Hoechst 33342 diluted in PBS (1:1,000) (10 mg/ml stock, Thermo Fisher) for 10 min at room temperature, protected from light. Slides were washed again in PBS and cover‐slipped with aqueous mounting medium.

An ImmEdge hydrophobic barrier pen (Vector Labs) was used in the following steps to create a hydrophobic barrier around the tissue sections to hold the reaction mix and wash solutions unless otherwise noted. The samples were washed three times with DEPC-PBS. The endogenous peroxidase activity was then blocked with 3% H₂O₂ by incubation at RT for 15 min, and the samples were washed three times with DEPC-PBS again. Next, a mixture containing 100 nM of each HR-HPV E6/E7 mRNA-targeting padlock probe (Table S1) in hybridization buffer (10% formamide in 6× SSC buffer [1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate]) was added to the sample and incubated at 37°C for 2 h, followed by three washes with 2× SSC containing 20% formamide for 5 min each to remove the unhybridized padlock probes. Circularization of the padlock probes was carried out by adding a ligation mixture containing 1× SplintR ligase reaction buffer (NEB), 50% glycerol, 0.5 U/μL SplintR DNA ligase (NEB), 1 U/μL RNase inhibitor (Thermo Scientific), and 0.2 μg/μL bovine serum albumin (BSA; NEB) to the sample and incubating it for 0.5 h at 37°C. After washing the sample three times with DEPC-PBST, 50 μL of 200-nM RCA primers (Table S1) in hybridization buffer (6× SSC buffer and 10% formamide) was added to the sample, which was then incubated at 37°C for 30 min. The sample was again washed three times with DEPC-PBST.

RNAscope Probes (Here we reported custom synthesized HvGAPDH and Rpg1 specific probes in barley, ACDBio™).
RNAscope V2 kit (ACDBio™).
Immedge^® Hydrophobic Barrier Pen (Vector Laboratories Cat. No. 310018).
Superfrost plus slides (ThermoFisher Scientific, 4951PLUS4).
Tissue-Tek^® manual slide staining set (Sakura, 4451).
Tissue-Tek^® 24-slide slide holder with detachable handle (Sakura, 4465).
HybEZ™ hybridization system and EZ-batch™ slide processing system (ACDBio™).
Humidifying paper (ACDBio™, 310025).
Incubator (VWR, 10055-006).
Water Bath (ThermoFisher Scientific, TSGP02).
Thermometer (VWR).
Zeiss LSM-700 confocal microscope.
Fresh 10% NBF (Millipore Sigma, HT501128-4L).
Ethanol (Millipore Sigma, E7023).
1 × PBS, Ph 7.4 (ThermoFisher Scientific, 10010023).
Everbright mounting media (Biotium, 23003).
Peel-A-Way embedding cryomold (Millipore Sigma, E6032-1CS).
Tissue- Tek^® optimal cutting temperature (OCT) compound (Sakura 4583).

Mice were anesthetized and perfused transcardially with PHEM buffer (1× PHEM: 60 mM PIPES, 25 mM HEPES, 5 mM EGTA, 1 mM MgSO₄; pH = 6.9) followed by ice-cold PHEM buffer containing 4% paraformaldehyde and 3.7% sucrose. Brains were dissected and post-fixed with perfusion buffer (4% paraformaldehyde, 3.7% sucrose, in PHEM buffer) for 2 hr at 4°C followed by washes with PBS and incubation with 30% sucrose in PBS at 4°C overnight. Next day, brains were embedded in NEG-50 frozen section medium following regular procedures. Sectioning was performed with a Leica cryostat at the coronal or sagittal plane at 30 μm thickness. Sections were placed on precleaned superfrost plus microscope slides (Fisher Scientific), left dry at room temperature and stored at –80°C until staining. For immunofluorescence, slides were sealed with ImmEdge hydrophobic barrier Pen (Vector Laboratories, Cat no. H-4000) and sections were blocked with 10% goat or donkey serum and 0.1% Triton X-100 in PBS for 1 hr at room temperature followed by incubation with primary antibodies diluted in 1% serum and 0.1% Triton X-100 in PBS, at 4°C overnight. Tissue was washed with PBS followed by incubation with secondary antibodies for 1 hr at room temperature. Finally, tissue was washed with PBS and covered with mounting medium (Vectashield) and a rectangular coverslip.

FFPE sections were sliced with 5 µm thickness from tissue samples and mounted on Superfrost plus (Thermo Fisher Scientific) slides. Slides were baked at 60°C for 2 h in a HybEZ™ II oven (Advanced Cell Diagnostics, catalog #321720) followed by deparaffinization with 100% ethanol and fresh xylene baths. Samples were pretreated with hydrogen peroxide for 10 min at RT, boiled with RNAscope antigen retrieval reagent (Advanced Cell Diagnostics, catalog #322000) for 15 min at 99°C, and then incubated with RNAscope protease plus reagent for 15 min at 40°C in the HybEZ™ II oven. Hydrophobic barrier was made around the tissue section using Immedge hydrophobic barrier pen (Vector Laboratories, Burlingame, CA).