Streptavidin alexa fluor 488

DNA extraction was performed using PureLink Genomic DNA Mini Kit (Invitrogen) following the manufacturer’s instructions. The probes were obtained from a PCR using genomic DNA of Peckoltia sp. 3 Jarumã and Peckoltia sp. 4 Caripetuba with primers previously described for 18S rDNA (Hatanaka and Galetti Jr, 2004 (link)), for 5S rDNA (Suarez et al., 2017 (link)) and U1 snDNA (Cabral-de Melo et al., 2012 (link)). These probes were labeled by nick-translation with biotin or digoxigenin. Telomeric probes were obtained from PCR using the set of primers F-5′(TTAGGG)₅-3′ and R-5′(CCCTAA)₅-3′ followed by labeling with Digoxigenin-11-dUTP (Roche Applied Science^®) (Ijdo et al., 1991 (link)). Fluorescence in situ hybridization (FISH) was performed as described by Martins and Galetti (1999) (link) using the following stringency conditions: 2.5 ng/μL of each probe, 50% formamide, 2 x SSC, 10% dextran sulfate, and hybridization at 42°C for 16 h. Fluorescent signals were detected using Streptavidin Alexa Fluor 488 (Molecular Probes, Carlsbad, CA, United States) and anti-digoxigenin rhodamine Fab fragments (Roche Applied Science, Penzberg, Germany). Chromosomes were counterstained with 0.2 μg/ml of 4′6-diamidino-2-phenylindole (DAPI) in Vectashield mounting medium (Vector, Burlingame, CA, United States).

Brain sections were deparaffinized, after antigen retrieval, in citrate buffer (10 mM sodium citrate, pH 6.0) for 30 min. The sections were pretreated with 0.3% H₂O₂ for 10 min to block endogenous peroxide activity after incubation with 6% normal serum and 0.1% Triton-X100 in PBS for 2 h. The sections were incubated with the following primary antibodies: anti-Cox2 (1:200, Abcam, Cambridge, UK) or anti-Iba1 (1:1,000, Wako Chemicals, Osaka, Japan). The samples were rinsed and incubated for 2 h at room temperature with secondary biotinylated anti-rabbit horse antibodies (1:200, Vector Laboratories, Burlingame, CA) followed by streptavidin alexa fluor 488 (1:200, Molecular Probes, Carlsbad, CA). Finally, the samples were mounted with Hard Set Vectashield with Dapi (Vector Laboratories, Burlingame, CA). Iba1 immunostaining was detected with a Zeiss 780 confocal laser scanning microscope. Cox-2 immunostaining was detected with a Zeiss Axio-photo 2 optic microscope. Cell counting was performed in three coronal sections for mouse at ×20 magnification.

The mean muscle fiber cross-sectional area was determined from transverse cryosections (6 μm) cut from the mid-belly of gastrocnemius muscles. Sections were labelled using immunohistochemistry to identify the basal lamina of muscle fibers. Sections were post-fixed in 10% (v/v) formalin for 5 min then washed and incubated with a rabbit polyclonal anti-laminin (#Z0097, DakoCytomation, 1∶100 in phosphate buffered saline (PBS) with 0.05% (v/v) Tween 20) overnight at 4°C. A biotinylated donkey anti-rabbit (#RPN1004, GE Healthcare Ltd, Auckland, NZ) secondary antibody was applied (1∶300) at room temperature for 30 min, followed by streptavidin Alexafluor 488 (Molecular Probes, Life Technologies NZ Ltd, Auckland, NZ) at 1∶400 for 30 min. To identify nuclei, DAPI (1∶1000 in PBS, 5 min) was used. Non-overlapping images of the entire muscle section were obtained using a microscope (DMI6000B) with motorized stage, digital color camera (DFC300) and capture software (AF6000) (Leica Microsystems, Wetzlar, Germany). Composite (tiled) images of the section were created and analysed. The mean fibre cross-sectional area was determined by manual tracing of the perimeter of clusters of contiguous fibers in artefact-free regions distributed across the section (mean  = 275 fibers per muscle) using commercial imaging software (ImagePro Plus, Media Cybernetics Inc, Rockville, MD).

Slide-mounted cryostat sections were blocked with BSA (5% in phosphate-buffered saline) for 1 h and then incubated with primary antibody singly or in combination overnight at 4°C in a humidified chamber. Different combinations of primary antibodies used were; 4G8 (Covance, 1/10000) alone for detection of β amyloid, 4G8 together with GFAP (Sigma Aldrich, 1/10000) for detection of reactive astrocytes, and 4G8 with biotinylated Isolectin GSA (Sigma Aldrich, 10 µg/ml) for detection of microglia. Post incubation, the sections were washed with PBS and probed with the secondary antibody/reagents as required, in a sequential manner. Fluorochromated secondary reagents used were Streptavidin AlexaFluor 488 (Molecular Probes, 1/200), Cy3Goat Anti Rabbit (Abcam, 1/200) and Streptavidin Cy3 (Jackson Laboratories, 1 µg/ml). Sections were examined by Axioplan2 (Carl Zeiss) and images captured by Spot RT-KE (Diagnostic Instruments).