Axioskop 2 microscope

Statistical analysis was performed with GraphPad Prism program (GraphPad Software, San Diego, CA). For the orofacial formalin test, the time spent (in seconds) in face rubbing was counted separately for Phase I and for Phase II. For mRNA expression, results were analyzed using the ΔCt method to compare expression of genes of interest with that of GAPDH. The area covered by CGRP and SP immunoreactive fibres in the TNC ipsilateral to the formalin injection, was expressed as optical density (OD) values, as suggested by previous reports [21 , 27 (link)]. OD was obtained using an AxioSkop 2 microscope (Zeiss) and a computerized image analysis system (AxioCam, Zeiss), equipped with dedicated software (AxioVision Rel 4.2, Zeiss, Germany). The mean OD was determined by rounding off the stained structure of interest (TNC) and subtracting the OD of the background (slide, mounting medium and coverslip) for each section, considering a total of 12 sections per animal. All sections were averaged and reported as the mean ± SEM of OD values.
All data were tested for normality using the Kolmogorov-Smirnov (K-S) normality test and considered normal. Differences between groups were analyzed by the one-way analysis of variance (ANOVA) followed by Tukey’s Multiple Comparison Test. A probability level of less than 5% was regarded as significant.

Cells were grown on culture slides. To remove soluble proteins and fix chromatin-bound proteins, cells were pre-extracted in buffer1 (1 % Triton X-100, 10 mM HEPES pH 7.4, 10 mM NaCl and 3 mM MgCl₂) supplemented with halt EDTA free protease and phosphatase inhibitors for 5 min at 4 °C. Cells were then fixed in 4 % paraformaldehyde for 10 min at 4 °C and then treated in buffer 2 (0.5 % Triton X-100, 20 mM HEPES pH 7.4, 50 mM NaCl, 3 mM MgCl₂ and 300 mM sucrose) supplemented with halt EDTA free protease and phosphatase inhibitors for 5 min at 4 °C. Cells were then blocked for 1 h in SuperBlock solution.
To look at Rad9 cellular localization cells were fixed with methanol and then rehydrated with PBS and blocked for 1 h in SuperBlock solution. Staining with primary antibody was performed overnight at 4 °C in blocking solution, whereas species specific fluorescein/Texas red conjugated secondary antibody (Vector Labs) was applied for 1 h at RT, followed by counterstaining with DAPI. All the primary antibodies were used at a 1:250 dilution, whereas the secondary antibodies were employed at a 1:500 dilution. Fluorescence images were captured using a Zeiss Axioskop 2 microscope.

Transverse EDL muscle sections were incubated for 3 min at room temperature in a sodium phosphate buffer containing 75 mM sodium succinate (Sigma), 1.1 mM Nitroblue Tetrazolium (Sigma) and 1.03 mM Phenazine Methosulphate (Sigma). Samples were then fixed in 10% formal-calcium and cleared in xylene prior to mounting with DPX mounting medium (Fisher). Densitometry of the samples was performed on a Zeiss Axioskop2 microscope mounted with an Axiocam HRc camera. Axiovision Rel. 4.8 software was used to capture the images.

SCs were plated on coverslips arranged in 24-well plates at the density of 2 × 10⁴ cells. Cells were pre-treated with 10 μM BK for 24 h. Then cells were treated with 10 μM ICH3 for 48 h. In the experiments with αBTX, this antagonist was supplied 2 h before ICH3. At the end of treatments, cells were washed 3 times with PBS and fixed with 4% paraformaldehyde in PBS for 20 min at RT. After 3 washes in PBS, cells were incubated with Phalloidin conjugated with Alexa Fluor™ 594 (Immunological Sciences, Milan, Italy) for 20 min to reveal actin filaments. After 3 washes in PBS, cells were incubated with Hoechst 33342 (1:1000 in PBS, Thermo Fisher Scientific, Waltham, MA, USA) for 10 min at RT, for the nuclei counterstaining. At the end, coverslips were fixed on microscope slides with a PBS-glycerol (3:1; v/v) solution. The images were acquired using an Axioskop 2 microscope (Zeiss, Oberkochen, Germany).

Morphometric and immunohistochemical analyses were performed in the four WAT depots from 14-month-old rats. Fragments of tissues were fixed in 4% paraformaldehyde in 0.1M phosphate buffer pH 7.3 overnight at 4°C, and then washed in the same buffer. For paraffin embedding, samples were dehydrated in ethanol, cleared in xylene, and embedded in paraffin blocks at 60°C.
For immunohistochemistry analysis, 5 μm sections were immunostained by means of the avidin-biotin technique using a commercial anti-MAC2 antibody (1:350 in PBS, Cederlane, Hornby, Ontario, Canada), counterstained with hematoxylin and mounted in Eukitt (Kindler, Freiburg, Germany). Images were acquired with a Zeiss Axioskop 2 microscope equipped with AxioCam ICc3 digital camera and AxioVision 40V 4.6.3.0 Software (Carl Zeiss, S.A., Barcelona, Spain). The software was also used to measure the diameter of 100 adipocytes (from one random field) in two non-consecutive hematoxylin/eosin stained sections. Image analysis from all groups was examined in a blind fashion.