Apotome 2 system

Paraffin sections from biopsy specimens from resistant/refractory MM patients (n = 3) and early‐stage MM patients (n = 4) were de‐paraffinized and rehydrated as previously described.³⁴ Sections were permeabilized using 0.3% Triton X and blocked to prevent non‐specific antibody binding using a 0.3% Triton X‐10% NGS solution. The slides were then incubated overnight at 4°C with the primary antibodies mouse anti‐MCT1 (Abcam) and rabbit anti‐CD138 (Abcam) at 1:100 dilution in 0.3% Triton‐X. Subsequently, cells were washed three times in PBS for 5 min and then incubated for 1 h at room temperature with the appropriate combination of fluorescence conjugated secondary antibodies donkey polyclonal anti‐rabbit Alexa Fluor 647 (A32849, Thermo Fisher Scientific; 1:500) and subsequently with goat polyclonal anti‐mouse Alexa Fluor 488 (A21247, Thermo Fisher Scientific 1:500). Samples were then washed in 0.3% Triton X in PBS and nuclei were counterstained with DAPI (1:1000) for 5 min, at room temperature. Slices were mounted with fluorescent mounting medium Permafluor (Thermo Fisher Scientific) and digital images were acquired using a Zeiss Axio Imager Z1 Microscope with Apotome 2 system (Zeiss, Milan, Italy).

Spleens were harvested and immediately fixed in 4% paraformaldehyde (PFA) in PBS for 3 h. Tissues were frozen-embedded in SubXero freezing medium (Mercedes Medical) and cut by cryostat microtome into 10-μm sections. To visualize reporters, sections were thawed in PBS, stained with Hoechst stain at a 1:10,000 dilution, and washed in PBS, and coverslips were mounted using ProLong Gold (Life Technologies). Tissue was imaged as described above, with an Apotome.2 system (Zeiss) for optical sectioning.

For the pharyngeal pumping rate, the number of grinder contractions was counted under a Zeiss Axiovert 25 microscope in 10-day-old adult animals fed different bacteria from embryo hatching. Ten worms were analyzed for each treatment, during a period of 30 s. The locomotion ability of nematodes was analyzed by body bending counting after 30 s. In particular, as described in [14 (link)], 10 worms for each treatment were washed in M9 buffer to remove bacteria, and then placed in 10 μL of M9 buffer to facilitate the locomotion measure. For lipofuscin accumulation analysis, 10-day-old adult worms, after washes in M9 buffer, were placed onto a 3% agar pad containing 20 mM of sodium azide. Afterwards, nematodes were observed with the Axio Observer Z1 inverted microscope, equipped with an ApoTome.2 System (Carl Zeiss Inc., Oberkochen, Germany). Digital images were acquired with the AxioCam MRm high-resolution digital camera (Zeiss) and processed with the AxioVision 4.8.2 software (Zeiss). ApoTome optical sectioning images of animals were recorded under a 40 Å~/0.75 objective (Zeiss). Median fluorescence intensity was analyzed using the ImageJ software, measuring the ratio of pixels per area of the worm.

Images were captured by an automated inverted fluorescence microscope with a structured illumination system (Zeiss Axio Observer Z1 with ApoTome.2 system) and Zen 2 (blue edition) software. The filter settings used were: FL Filter Set 34 (Ex. 390/22, Em. 460/50 nm), Set 38 HE (Ex. 470/40, Em. 525/50 nm), Set 43 HE (Ex. 550/25, Em. 605/70 nm), Set 50 (Ex. 640/30, Em. 690/50 nm), and Set 63 HE (Ex. 572/25, Em. 629/62 nm). The objectives used were: Fluar 2.5x/0.12, EC Plan-Neofluar 5x/0.16, Plan-Apochromat 10x/0.45, EC Plan-Neofluar 20x/0.50, EC Plan-Neofluar 40x/0.75, Plan-Apochromat 63x/1.40. Images were typically tile-scanned with a motorized stage, Z-stacked, and reconstructed by a maximum intensity projection (MIP) function. Differential interference contrast (DIC) was used for objectives higher than 10×. Representative images of at least three independent biological samples are shown in the figures. Quantification of cells on sections was performed using NIH Image J software.

For immunofluorescence detection, U2OS cells were grown on coverslips in six-well plates. The cells were fixed using 3.3% paraformaldehyde (PFA) in DMEM with 10% FBS for 20 min, washed in phosphate-buffered saline (PBS), and permeabilized for 15 min with 0.5% Triton X-100 (TX-100) in PBS with 10% FBS. The cells were then incubated for 1 h with primary antibodies diluted in PBS with 10% FBS (see Table S1 for the antibody list). After washing, the cells were incubated for 45 min with the corresponding secondary antibodies labelled with Alexa Fluor dyes diluted in PBS with 10% FBS (1:1000; Invitrogen). After the final incubation and washing, coverslips were mounted using ProLong™ Gold Antifade reagent with DAPI (Invitrogen; P36935). Images were acquired using a Zeiss apotome.2 system in apotome mode with 2776×2080 co-site sampling using an Axiocam HRm camera on an Axio Observer Z.1 with Colibri-led illumination with LED wavelengths (365 nm, 470 nm, 555 nm and 625 nm) and appropriate emission filters using the 63× oil immersion objective. Image pixel size is given to be 0.05×0.05 µm. Images were processed using the ImageJ software (Schneider et al., 2012 (link)). To compare control cells with cells treated with chemicals or siRNA, images for each channel were acquired with identical illumination and exposure settings and processed identically.

Apoptosis was evaluated using the In Situ Cell Death Detection Kit, Fluorescein (Roche, Lewes, UK) in the spheroids obtained in SMG. Spheroids were fixed in PFA 4%, embedded in Technovit 8100 resin (BioOptica, Milan, Italy) and sectioned. Sections were permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate and exposed to TUNEL reaction mixture for 1 h at 37 °C, in a humidified atmosphere, protected from light. After PBS washes, sections were analyzed with AxioObserver Z2 inverted microscope (Zeiss), with Apotome2 system (Zeiss) and ZEN Blue 2.6 image acquisition software (Zeiss).

Immunofluorescent analysis was performed as already described [21 (link)]. After treatment, cells were adhered to slides by cytospin and subsequently fixed with 4% formaldehyde for 20 min at room temperature. The slides were then incubated overnight at 4 °C with the primary antibody against Nrf2 (anti-rabbit; Santa Cruz Biotechnology, Dallas, TX, USA) and NF-kB (anti-rabbit; Santa Cruz Biotechnology) at a dilution of 1:100. The slides were mounted with medium containing DAPI (4,6-diamidino-2-phenylindole) to visualize nuclei.
To investigate mitophagy, cells were labeled with 200 nM MitoTracker Red CMXRos probe (M7512, Thermo Fisher Scientific, Rodano, Milan, Italy) before the treatment. Cells were subsequently incubated with primary antibody against LC3-II-rabbit (L7543, Sigma-Aldrich, Milan, Italy) at 1:100 dilution. The fluorescent images were obtained using a Zeiss Axio Imager Z1 Microscope with Apotome 2 system (Zeiss, Milan, Italy).