Slowfade gold antifade mountant with 4 6 diamidino 2 phenylindole dapi

Transfected HeLa cells were seeded onto 15 mm circular glass coverslips (Matsunami, C015001) in a 12-well plate at 1×10⁵ cells per well to ensure ∼80% confluency the following day. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, blocked with 20% goat head serum in PBB (0.5% BSA in PBS) and incubated with one of the following primary antibodies overnight at 4°C at a concentration of 1:100: mouse anti-Nrf2 (Abcam, ab62352), mouse anti-Keap1 (Proteintech, 10503-2-AP) or rabbit anti-Hsp90 (Proteintech, 13171-1-AP). Coverslips were incubated with the following Alexa Fluor 680-conjugated secondary antibody for 1 h at room temperature at a concentration of 1:1500: goat anti-mouse (Thermo Fisher Scientific, A-11094) or goat anti-rabbit (Thermo Fisher Scientific, A11036). Coverslips were then mounted onto glass microscope slides with SlowFade Gold Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific, S36938) and cured at room temperature for 24 h. Cells were imaged using the Cytation 5 Cell Imaging Multi-Mode Reader (BioTek) using a 20× objective lens and captured using Gen5 Software (BioTek).

A probe, MelTpq-646 (Table S2), specific to the 16S rRNA of phylotype Tpq-Mel-01, was designed using ARB and labeled with Texas Red at the 5′ end. The probe has at least three mismatches against sequences in the SILVA database. The entire guts were removed from two T. propinquus workers, and gut contents were suspended in solution U and incubated at room temperature for 30 min in order to precipitate and remove large particles. The supernatant was collected into a new tube and fixed with 4% paraformaldehyde at 4°C for 5 h. The mixture was centrifuged at 12,000×g for 10 min, and the pellet was washed three times with sterile DDW during centrifugation. The pellet was resuspended in 100 μL DDW, spotted onto MAS-GP slide glasses (Matsunami, Osaka, Japan), and air-dried. Subsequent procedures were as described previously (27 (link)) with hybridization at 55°C for 1 h. Specimens were enclosed with the SlowFade™ Gold Antifade Mountant with 4,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific). Observations were conducted under an Olympus BX51 epifluorescence microscope (Olympus, Tokyo, Japan). Cell sizes were measured with ImageJ software (https://imagej.nih.gov/ij/).

In order to detect Arcicella cells associated with ice worms, an Arcicella-specific probe, Arci_1251 (5′-GTGTTACCACATAGCG ACCTGC-3′), which corresponds to positions 1251–1272 in E. coli (J01695), was designed using ARB and labeled with Texas Red at the 5′ end. A non-labeled helper probe (5′-GGTTTTGTAGATT GGCACT-3′) was used to improve the efficiency of hybridization (16 (link)). The preparation of 10-μm-thick cryosections of ice worms was conducted using a Leica CM 1850 cryostat (Leica Biosystems, Nußloch, Germany). Hybridization was performed at 55°C for 2 h, and specimens were enclosed using the SlowFade^® Gold Antifade Mountant with 4,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, Waltham, MA, USA). Specimens were observed under an Olympus BX51 epifluorescence microscope (Olympus, Tokyo, Japan). Procedures were described in detail previously (27 (link)).