Dm5000b fluorescent microscope

The cells of snakes and frogs were obtained from tissues of each individual and fixed in Carnoy fixative (methanol:acetic acids = 3:1 mixture). The slide spreads were prepared according to the standard procedure and FISH was carried out as described previously (Tanabe et al. 1996 (link), 2021 (link)). The BovB_VA sequence cloned in pUC57 (total 5,879 bp) were labeled by nick translation with digoxigenin-11-dUTP (Roche 11093088910) and used as probes. Since BovB_VA could not hybridize with bufonid BovBs due to low sequence similarity (<80%), we made a specific probe for bufonids. Specifically, BovB amplicon from B. japonicus was cloned in pCR2.1-TOPO (total ca. 6,700 bp) and used as the probe. Labeled DNA probes were hybridized for 24–36 h onto slide spreads. After hybridization, the slides were washed and detected with mouse antidigoxigenin antibody (Sigma D-8156) in the first layer and successively detected with sheep antimouse, Cy3-conjugated antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) in the second layer. The slides were counterstained with DAPI and mounted in Vectashield Antifade (Vector Laboratories, Inc., Burlingame, CA). FISH images were captured and analyzed using a Leica DM5000B fluorescent microscope equipped with a CCD camera and CW4000 image analysis software (Leica Microsystems, Wetzlar, Germany).

HLE and Huh7 cells following different treatments were seeded in confocal dishes (NEST, VA, USA) and maintained in DMEM for 24 h. The cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100, and then incubated with primary antibodies overnight at 4 ˚C. Antibodies were used as follows: anti-ZNF281 (1:50, Santa Cruz, CA, USA), anti-TFAM (1:50, Cell Signaling Technology Inc., Danvers, MA, USA), anti-NRF1 (1:50, Cell Signaling Technology Inc., Danvers, MA, USA), anti-PGC-1α (1:50, Millipore, Billerica, MA, USA), anti-SDHB (1:200, Abcam, Cambridge, MA, USA), and anti-MT-CO2 (1:200, Abcam, Cambridge, MA, USA). The fluorescent secondary antibodies used were Alexa fluor (m) 594 goat A or Alexa fluor (R) 488 goat A (1:200, Invitrogen, NY, USA). Counter-staining of the nuclei was performed using 4′, 6-diamidino-2-phenylindole (DAPI). Samples were observed under a Leica DM5000 B fluorescent microscope and Leica SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany).

Spleens were snap frozen in OCT compound and cryosections (5–6 microns) were prepared as described 43 (link). Immunohistology was performed using the Abs listed above. For detecting FDCs a three-step approach was used. Sections were first stained with rat anti-mouse FDC (FDC-M1), then a biotinylated anti rat IgG was used as secondary Ab, and a SA-APC conjugate was used to detect biotin. Stained sections were analyzed using a Leica DM5000B fluorescent microscope (Leica microsystems, Germany) and captured digital images were processed using the Leica Application Suite (LAS) imaging software.

In order to generate a C-terminal GFP-tagged SVR11, the coding sequences of SVR11 amplified with primers 20360F and 20360 GFPR, SVR11-like with 02930F and 02930 GFPR, were cloned into transient expression vector pTF486. The resulting construct were designated p35S::SVR11-GFP and p35S::SVR11-like-GFP. Arabidopsis leaf protoplast preparation and transient expression of GFP constructs were performed as described by Yoo et al. (2007) (link). Bright field images and fluorescent signals from GFP and chlorophyll autofluorescence were monitored using a Leica DM5000B fluorescent microscope (Leica, Germany).