Fig. 4

Immunofluorescence visualization of i-motif structure in the B. mori testis. a Testes were isolated from the 5th instar larvae and treated as described in “Materials and Methods” section. b Schematic diagram of i-motif immunofluorescence detection. The primary antibody was an anti-BmILF antibody at a 1:2000 dilution; the secondary antibody was Alexa Fluor™ 594-conjugated goat anti-rabbit IgG at a 1:400 dilution. The red fluorescence shows the binding of BmILF to the i-motif structure in the nuclei of testis cells. c BmILF antibody recognizes the endogenous BmILF-bound i-motif structure in the nucleus, without the addition of exogenous BmILF. d Increased i-motif staining after incubation with exogenous recombinant BmILF. e Immunofluorescence staining of the i-motif after treatment with DNase I. The dotted lines show the boundaries of the nuclei; f the white light and fluorescence merged image of e, showing the position of the nuclei. g Immunofluorescence staining of the i-motif after the pre-incubation of BmILF with pre-folded C-quadruplex oligonucleotides. Nuclei were counterstained with DAPI (blue). h Quantification of i-motif signals for 50 nuclei in five random observation regions on the slide. The data are represented as the mean ± SEM of three replicates