Mbd0015

Fixed cultures were permeabilized for 1 h in blocking solution [3% BSA (w/v), 10% normal goat serum (v/v), 0.2 M glycine in PBS, 0.1% Triton-X100 (v/v)] (Kontou et al., 2021 (link)). Coverslips were incubated with primary antibodies diluted in blocking solution for 1 h at room temperature. After a brief wash with PBS, the coverslips were incubated with fluorophore-conjugated secondary antibodies diluted in blocking solution for 1 h at room temperature. The coverslips were then washed in PBS, briefly dipped in DAPI ready-made solution (Sigma-Aldrich MBD0015), and mounted onto microscope slides with Fluoromount-G (SouthernBiotech 0100-01). The coverslips were imaged using a Nikon A1 confocal microscope (Nikon Instruments, Melville, NY, USA) using a 60× oil immersion objective lens. Laser settings were manually assigned for each fluorescent channel and images were acquired at the scan size of 1,024 × 1,024. Settings were kept the same between imaging sessions and between genotypes.

De-paraffinized heart sections or HUVECs on coverslips were stained with primary antibodies overnight at 4°C, and then secondary antibodies conjugated with fluorescent-dye for 2 h at room temperature. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI, MBD0015, Sigma-Aldrich). Fluorescence images were captured by Leica TCS SP8 confocal fluorescence microscopy.

hiPSCs, hiPSC-derived NPCs and neurons cultured on coverslips were fixed with 4% paraformaldehyde (PFA) for 15 min. Subsequently, the cells were permeabilized using 0.3% Triton X-100 and blocked with a solution containing 3% normal donkey serum and 0.1% Triton X-100 in PBS. The cells were then incubated with primary antibodies, including anti-OCT4 (sc-5279, Santa Cruz, 1:500), anti-NESTIN (MAB5326, Millipore, 1:200), anti-PAX6 (PRB-78P, Convance, 1:300), anti-MAP2 (MAP3418, Millipore, 1:200), anti-DARPP-32 (sc-11365, Santa Cruz, 1:100), and anti-SYP (ab68851, Abcam, 1:100), overnight at 4 °C. Following this, the appropriate Alexa Fluor secondary antibodies were applied for 1 h at room temperature, followed by staining with 1 mg/mL DAPI (MBD0015, Sigma-Aldrich, USA) for 10 min. The images were acquired using an Olympus FV100 inverted confocal microscope.

Samples were fixed in 4% paraformaldehyde solution in 0.1 M phosphate-buffered saline (PBS) for 8 h at 4 °C, then washed in 0.1 M PBS, incubated in 5% Triton X-100 solution in PBS for 24 h, and blocked in 1% solution of bovine serum albumin in PBS for 6 h. The blocked specimens were incubated in mixture of rabbit anti-5-HT (S5545, diluted 1:1000; Sigma-Aldrich, St. Louis, USA) and rabbit anti-FMRFamide antibodies (AB15348, diluted 1:1000; EMD Millipore, Burlington, Massachusetts, USA). Rediae were incubated in primary antibody for 24 h, then washed in PBS with 0.1% Triton X-100 and incubated in the secondary anti-rabbit CF488 antibody (SAB4600044, diluted 1:1000; Sigma-Aldrich) for 8 h. After antibody incubations samples were washed in PBS, and then incubated in DAPI staining solution (MBD0015, diluted 1:1000; Sigma-Aldrich) for 5 min, washed in PBS and mounted in glycerol. The specimens were examined under Leica TCS SP5 MP confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany) and analyzed using Fiji software [59 (link)].

Fresh surgical cancer resections from the oral cavity were imaged to evaluate the imaging performance of DeepDOF-SE. In our ex vivo protocol, consenting patients undergoing surgery for oral cancer resection were enrolled. The excised specimen was first assessed by an expert pathologist and sliced into 3–4-mm-thick slices with a standard scalpel. Selected slices were processed for standard frozen-section pathology. The remaining slices were cleaned with phosphate-buffered saline (PBS, Sigma-Aldrich P4417, pH 7.2–7.6, isotonic) to remove residuals such as mucus and blood, stained with DAPI (Sigma-Aldrich MBD0015, diluted with PBS, 500 μg/mL) for 2 min and Rhodamine B (Sigma-Aldrich 83689, dissolved in PBS, 500 μg/mL) for 2 min, and excessive stain was rinsed off with PBS. The tissue was then imaged using the DeepDOF-SE microscope. The raw frames were processed with the DeepDOF-SE networks and stitched using Image Composite Editor (Microsoft, discontinued and other stitching software can be applicable). Post-imaging, the specimens were processed through FFPE histopathology at University of Texas MD Anderson Cancer Center, and the slides were imaged using a slide scanner to provide the standard H&E images. The study was approved by the Institutional Review Boards at the University of Texas MD Anderson Cancer Center and Rice University.

To release the meiocytes, 4% PFA-fixed anthers were squashed in distilled water using a needle on a MAS-coated microscope slide, and incubated at 25 °C for 30 min. The meiocytes were then blocked with 3% BSA in PME buffer (50 mM PIPES, 5 mM EGTA, and 5 mM MgSO₄; pH 6.9) for 60 min and incubated at 4 °C overnight with rabbit anti-AGO1b, mouse anti-AGO1d, and guinea pig anti-MEL1 antibody, diluted 1/500 with 3% BSA in PME. After washing three times with PME for 5 min, the slide was incubated in a dark chamber for 3 h at 25 °C with Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 (Invitrogen, A11036), Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Invitrogen, A11001), and Goat anti-Guinea pig IgG HL, Alexa Fluor 647 (abcam, ab150187), diluted 1/200 with 3% BSA in PME, followed by three 5-min washes with PME. The samples were incubated for 15 min at 25 °C in 1 µg/mL DAPI diluted 1/1000 with PME (Sigma, MBD0015), followed by washing three times with PME buffer. Samples were mounted in ProLong Gold antifade reagent (Invitrogen, P10144) and images were captured using an LSM 880 microscope (Carl Zeiss)^{31 (link)}.

Organoid sections were washed three times in 0.2% Triton-X in PBS (0.2%PBS/T) and then blocked in 10% normal goat serum diluted in 0.2%PBS/T (blocking solution) for 1 h at room temperature. Sections were incubated in primary antibodies diluted in blocking solution overnight at 4 °C and were subsequently washed three times with 0.2%PBS/T, followed by incubation with secondary antibodies and DAPI diluted in PBS/T at room temperature for 2 h. Finally, sections were washed three times (20 min per wash), and then mounted using Fluoromount mounting media (Southern Biotech). Primary antibodies used are as follows: mouse anti-Ki67 (1:250, BD Biosciences BDB550609), rabbit anti-SOX2 (1:500 Cell Signaling 3697S), rabbit anti-HOPX (1:500, Proteintech 11419-1-H), rat anti-CTIP2 (1:2000, Abcam ab18465), rabbit anti-TBR2 (1:1000, Abcam ab23345), mouse anti-SATB2 (1:1000 Abcam ab51502), mouse anti-NEUN (1:500, EMD Millipore MAB377), rabbit anti-NEUN (1:1000, EMD Milipore ABN78), rabbit anti-S100B (1:1000, Sigma S2644), chicken anti-MAP2 (1:5000, Abcam ab5392), rabbit anti-SYNAPTOPHYSIN (1:1000, Abcam ab14692), rabbit anti-HOMER (1:1000 Synaptic System 160003), and mouse anti-SYNAPSIN (1:500, Synaptic System 111011). Secondary antibodies conjugated with Alexa 488, 594, 647 (Invitrogen) and DAPI (1:1000, Sigma MBD0015) were used.