4 6 diamidino 2 phenylindole dihydrochloride dapi

Whole brains were sectioned (12 µm coronal) on a cryostat (CM 1900, Leica), and mounted on glass slides. Slide-mounted sections were fixed using 4% paraformaldehyde (PFA), then permeabilized and blocked in bovine serum albumin followed by incubation with antibodies against glial fibrillary acidic protein (GFAP) (1:400; Sigma), and ionized calcium binding adaptor molecule 1 (IBA1) (1:400; GeneTex) overnight at 4°C. Sections were washed and then incubated with rabbit polyclonal secondary antibodies coupled to AlexaFluor 488 or AlexaFluor 568 (Invitrogen). To confirm specificity, additional sections were incubated without the primary antibody. Nuclei were labelled by staining with DAPI (4′,6′-diamidino-2-phenylindole dihydrochloride; Vector Laboratories), and sections were examined using a Leica DM4000 epifluorescence microscope. Counts of GFAP+ and IBA1+ cells were performed blind to treatment. One dorsal (−2.8 AP) and one ventral (−2.2 AP) representative region within each hippocampal subfield was selected, and cells were counted under ×20 lens magnification. Representative images of the GFAP/IBA1 staining were taken using the same exposure time without knowledge of the treatment group. We performed an additional analysis of the microglia in the CA3 subfield, by assessing the number and length of processes, as described (Young and Morrison, 2018 (link)).

Nuclei and kinetoplasts were visualized by fluorescence microscopy as described [24 (link)]. Briefly, trypanosome cultures were adjusted to 5 × 10⁵ cells/ml in HMI-9 and 50 μl was spread out on a microscope slide and allowed to air dry. The slides were placed in ice-cold methanol overnight at -20°C and subsequently dried at room temperature. The preparation was rehydrated for 5 min with 1 ml PBS, which was removed by tipping the slide to its side prior to the addition of 20 μl Vectashield mounting medium containing 1 μg/ml DAPI (4′,6-Diamidino-2-phenylindole dihydrochloride; Vector Laboratories, Burlingame, CA), before covering with a cover slip. The slide was then viewed using a DeltaVision fluorescence microscope.

Deparaffinized 3 μm thick sections were pretreated for 20 min with 0.2 M HCl, incubated with 1 M NaSCN for 30 min at 80°C, washed in dH₂O and 2x SCC and digested with 1 mg/ml pepsin (2,500–3,500 U/mg, Sigma Chemical, St. Louis, MO) in 0.14 M NaCl solution, pH2. The biotin labeled full length MCPyV DNA probe was added to the samples at a concentration of 5 ng/μl followed by denaturation of DNA (5 min, 80°C) and hybridization overnight (37°C, humid chamber, Thermobrite, Abbott, IL). Unbound MCPyV DNA probe was stringently washed away in 2x SSC, pH7 at 70°C for 2 min. Bound probe was detected by sequential incubation in a combination of fluorescein isothiocyanate (FITC) biotinylated avidin (AvFITC; 1:500; Vector, Brunswig Chemie, Amsterdam, The Netherlands) and biotin conjugated goat antiavidin (BioGaA; 1:100; Vector). Prior to incubation aspecific binding sites were blocked with Boehringer Blocking reagent. Cell nuclei were counterstained and coverslipped with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI; 0.2 μg/ml, Vectashield, Vector Laboratories, CA). Samples were visualized using a DM 5000B fluorescence microscope (Leica, Wetzlar, Germany) coupled to an online digital camera (Leica DC 300 Fx) for independent evaluation of FISH signals by 3 investigators (AzH, DR, LH) according to criteria described earlier (Hafkamp et al., 2008 (link); Haugg et al., 2014 (link)).

Following 5 days of cell differentiation, the medium was removed and cells were fixed in 90% (v/v) methanol in TBS at −20 °C for 30 minutes^{62 (link)}. SH-SY5Y and U-87MG cells were stained for βIII tubulin and glial fibrillary acidic protein (GFAP), respectively. Cells were permeabilised with 1% (v/v) Triton X-100 in TBS. Primary mouse monoclonal anti-βIII tubulin (1:2000; Sigma-Aldrich) and rabbit polyclonal anti-GFAP (1:500; New England Biolabs, UK) antibodies were diluted in 3% (w/v) bovine serum albumin in TBS and incubated at 4 °C overnight. Because PAN and PJ nanofibres autofluoresce emitting green light, Alexa Fluor^™ 568 goat anti-mouse Ig for anti-βIII tubulin and Alexa Fluor™ 568 chicken anti-rabbit Ig for GFAP (both from Cell Signalling, UK) were used at 1:200 dilutions. Cells were mounted on glass slides and counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Vector Laboratories Ltd., UK).

The cells were fixed for 20 min with 4% paraformaldehyde and 0.1% Triton X-100 in PBS buffer. After washing with PBS, the fixed cells were incubated in a blocking solution (PBS containing 0.5% FBS) for 1 h at room temperature to reduce nonspecific antibody binding. The cells were treated with diluted primary antibodies in a blocking solution for 1 h at room temperature or overnight at 4 °C. After washing three times with PBS, the cells were incubated with Alexa Fluor-conjugated secondary antibodies (1000:1). The nuclei were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Vector Laboratories; Burlingame, CA, USA; H-1200). The cells were imaged by using a Nikon Eclipse TE2000 fluorescence microscope. As indicated in the text, the neuronal cells were also imaged using confocal microscopy (LSM 510 Meta and 800; Zeiss, Göttingen, Germany) with a × 2 × , × 40, and × 100 objective, and the images were printed. The antibodies used for immunocytochemistry are listed in Supplementary Table 4.