Lsm 880 airyscan confocal microscope

10 to 12 week old tumors were dissected and fixed in 10% neutral buffered formalin overnight at room temperature. They were then embedded in paraffin and sectioned in 5–10 um sections. In some cases, sections were stained with H and E, picosirius red, or trichrome stain prior to SHG imaging. Prior staining had no effect on SHG signal. Images were acquired on a Zeiss LSM 880 Airyscan confocal microscope using an inverted, motorized Zeiss Axio Observer Z1 frame. Two-photon images were collected at 880 nm, using non-descanned detectors set to 440 nm for SHG. Three to four z-stacks were acquired (step size 2 um) per tumor. The z-stacks were compressed and TACS signature was scored by three blinded reviewers as previously described (Corsa et al., 2016 (link)) (Provenzano et al., 2006 (link)).

Embedded bones were sectioned at 100 µm thickness using a cryostat from Leica (CM3050 S). Sections were hydrated using PBS and permeabilized using 0.5% Triton-X100 in PBS for 15 min at room temperature (RT). Samples were then incubated in blocking solution (0.3% Triton, 1% BSA, 2% donkey serum in PBS) for 30 min at RT prior to primary antibody staining. Primary antibodies were diluted in blocking solution, incubated O/N at 4 °C. Species-specific secondary antibodies (Jackson) diluted in blocking buffer were added and incubated for 2–3 h at RT. Nuclei were stained with Hoechst (1/1000 in PBS) and mounted using FluorSave mounting media. The following primary antibodies were used: rat monoclonal anti-Endomucin (sc-65495, Santa Cruz, diluted 1/200), goat polyclonal anti-CD31 (AF3628, R&D, diluted 1/100), rat monoclonal CD41 FITC‐conjugated (MWReg30, Biolegend, diluted 1/50). All secondary antibodies were from Jackson ImmunoResearch and diluted 1/200.
Images were acquired using a LSM 880 Airyscan Confocal microscope (Zeiss), and processed and analyzed using Zen (Zeiss) and Fiji softwares.

The ability of Der p 1 reactive membrane antibodies to internalize proDer p 1 fusion proteins after binding to Der p 1 reactive hybridoma cell lines was confirmed by confocal microscopy. Briefly, 96-well flat bottom microscopy plates (Ibidi, Germany) were plasma-treated with fibronectin (Thermo fisher scientific) to promote cell attachment. About 5 × 10⁵ cells (4C1/ 10BP) were plated and incubated with complete medium overnight. After 24 h, cells were washed with sterile 1× PBS (pH 7.4) before staining with 20 μg of Alexa 647 labelled proDer p 1-SNAP or proDer p 1-ETAʹ fusion proteins for 1 h. In some instances, labelling of cells was carried out on ice (to reduce cell metabolism and by so doing prevent internalization of fusion proteins). After staining, the cells were washed twice with 1× PBS (pH 7.4) and counterstained with the nuclear stain; DAPI. Next, cells were washed and fixed with 2% paraformaldehyde. Images were taken using the LSM 880 Airyscan confocal microscope (Zeiss) at 40× magnification.

Vibratome sections (Leica VT1000S) (30 μm) were washed 3 times in PBS, blocked in 10% normal goat serum, 1% bovine serum albumin, 100 mM glycine, 0.1% Triton-X, in PBS for 1 h at room temperature and incubated with primary antibodies overnight at 4 °C in 1% normal goat serum, 1% bovine serum albumin, 0.1% Triton-X, in PBS. Samples were then washed in PBS and incubated in secondary antibodies for 1 h at room temperature. Primary antibodies used were: guinea pig anti-DCX (1:2000, Millipore, AB2253), rabbit anti-GFAP (1:1000, Daco, Z0334), rat anti-BrdU (1:1000, Bio-Rad, OBT0030G), mouse anti-L1cam (1:300, Abcam, [2C2] ab24345), rabbit anti-Sox2, (Millipore, ab5603) (1:2000), mouse anti-PCNA (Abcam, ab29) (1:500). PCNA was detected with antigen retrieval in sodium citrate buffer (10 mM Sodium Citrate, 0.05% Tween 20, pH 6.0) at 95 °C hot plate for 45 min, cooled down for 20 min, and washed 2x in PBS for 5 min. Secondary antibodies used were goat anti-mouse, -rabbit, -guinea pig, and -rat Alexa Fluor 488, 568, and 647. For BrdU staining, before the blocking step, sections were incubated in 2 M HCl at 37 °C for 30 min, followed by 0.1 M sodium borate, pH 8.5 for 20 min, and PBS. Sections were imaged using a Zeiss AxioSkop2 or a confocal Zeiss LSM880 Airyscan Confocal Microscope.

Transduced cortical neurons were transferred to chambers containing pre-warmed imaging buffer (Hibernate E low fluorescence medium (BrainBits) with 2% B27 and 0.5 mM GlutaMAX). A Zeiss LSM 880 Airyscan confocal microscope with a 40 × 1.3 NA oil immersion objective was used for live neuron imaging. To assess cellular ATP levels, images were collected at emissions 505–550 nm and above 545 nm within cell bodies or axons expressing GO-ATeam2 to measure ratiometric values which were generated in ImageJ (Huang et al. 2021 (link)). To analyze axonal mitochondrial transport, live images were captured along the microgrooves with 5-sec intervals, and kymographs were generated using ImageJ (Kang et al. 2008 (link)).