Axiocam erc 5s

Retroperitoneal adipose tissue was included in Tissue-Tek^® medium, frozen in liquid nitrogen and 5 μm sections were obtained and adhered onto silanized slides. Slides were stained with HE. Evaluation of leukocyte infiltrate was also performed by immunohistochemistry. Slides were blocked with 3% BSA and incubated overnight with mouse anti-rat CD11b/c mouse antibody (BD Pharmingen ^™, 1:1000). Subsequently, the tissue was incubated with biotinylated anti-mouse IgG (Jackson ImmunoResearch, 1:200) secondary antibody for 2 hours, washed with 0.1M phosphate buffer and incubated with VectaStain ABC kit (Vector Laboratories, 1:100) for 2 hours. Detection of the antigen-antibody complex was performed through the chromogen 3,3'-diaminobenzidine (DAB) for 5 minutes at room temperature. Sections without the primary antibody (Cd11b/c) were used as negative control of the immunolabeling process. The qualitative evaluation of the slides was performed using photomicrographs captured by Zeiss Axiovert 40 microscope, with a 40x objective (Camera: Zeiss AxioCam ERc 5s).

Bioluminescent validation of localized inflammation was performed by evaluating for myeloperoxidase activity in select mice using an i.p. administration of luminol (XenoLight RediJect Chemiluminescent Inflammation Probe, PerkinElmer, Waltham, MA). 100 µL of luminol was injected 24-hours post LPS-induced myositis, and bioluminescence was measured 10-min after the i.p. injection, using an IVIS Lumina system with a five-minute exposure time and medium binning. The bioluminescent images were analyzed with Fiji 101 (link) and co-registered with an X-ray projection image, taken with XPERT cabinet X-ray system (KUB Technologies, Inc.), as anatomical reference. Tissue were harvested after euthanization and fixed in 10% formalin. Fixed tissue sample was stained for Prussian blue, hematoxylin and eosin (H&E), and myeloperoxidase (LifeSpanBio Sciences, Inc.). Bright field images of the slides were observed using a ZEISS AX10 Observer D1 with a ZEISS Axiocam ERc 5s (ZEISS Microscopy, Germany).
All statistical analysis was carried out using standard student t-test of the means.

After collection and pre-fixation in 4% buffered paraformaldehyde, liver from the three experimental groups were dehydrated by a growing series of alcohols, diaphanized in xylol, embedded in paraffin and 4 μm liver sections were obtained and mounted onto silanized slides. Sections were stained by Hematoxylin & Eosin (HE) technique and used in the morphometric and quantitative evaluation of liver cells. The area (μm²) of the hepatocytes’ cellular and nuclear profiles were determined by measuring 50 cells and nuclei/animal, randomly selected, summing 250 cells/group. Cell density (cells/mm²) was determined as described by Mandarin-de-Lacerda [47 ], using 5 semi-serial sections/animal and 2 fields/section were analyzed, totalizing 10 photomicrographs/animal. Morpho-quantitative analyzes were performed using a computerized imaging device (Axio Vision 4.5 Zeiss ^®) coupled to a 40x objective trinocular microscope (Zeiss Axiovert 40; Camera: Zeiss AxioCam ERc 5s).

Cell proliferation was evaluated by average size and number of 3D tumor colonies under anchorage-independent growth conditions in 5 mg/mL Matrigel (MG; Corning Incorporated, NY, USA). Therefore, as explained above, a 96-well plate (Sarstedt AG & Co. KG, Nümbrecht, Germany) was coated with MG diluted in the appropriated culture medium. After solidifying at 37 °C, the upper layer of an MG cell suspension (38 cells/well, in a final volume of 53 µL cell MG suspension each well) was plated as described above. The plate was then incubated for 45 min at 37 °C and finally augmented with the adequate culture medium. The colony formation took 11 days of incubation at 37 °C and 5% CO₂. A change in the medium was performed after 7 days. For the evaluation of colony number and surface area, images were captured using Zeiss Axiovert 35 and Zeiss Axiocam ERc 5s (Carl Zeiss, Jena, Germany) of colony number and surface area. ZEISS ZEN lite 3.5 (blue version) software (Carl Zeiss Microscopy, Oberkochen, Germany) was used to photograph the colonies at 10× magnification. The colony area was determined in µm² by ImageJ 1.53 (NIH, NY, USA).

Liver was included in Tissue-Tek^® medium, frozen in liquid nitrogen and 10 μm sections were obtained and adhered onto silanized slides. Slides were fixed in 4% buffered paraformaldehyde, washed in distilled water and stained with Oil Red O and hematoxylin for lipid analysis. The qualitative evaluation of the slides was performed using photomicrographs captured by Zeiss Axiovert 40 microscope, with a 40x objective (Camera: Zeiss AxioCam ERc 5s).

Ovarian tumor tissue was fixed in a neutral (buffered) 4% formaldehyde solution for 24 h, dehydrated and saturated with paraffin. Paraffin blocks were sectioned with a thickness of 4 μm with a Shandon Finesse 325 rotary microtome (Thermo Scientific, Waltham, MA, USA). After deparaffinization and dehydration (with xylene and ethanol), histological sections were stained with hematoxylin and eosin. All photos were captured with a digital visualization system based on a Zeiss Primo Star microscope with a Zeiss Axiocam ERc 5s digital camera and software package “Zen 2.0” (Carl Zeiss, Jena, Germany).

The materials for morphological studies were the samples of biopsy tissues excised from the mucous membrane in the area of immediate localization of the pathological process in all patients with HCV. According to the standard histological scheme, the pieces of the tissue were fixed in 10% neutral formalin, dehydrated and embedded in paraffin. A series of sections 4 µm in thickness were stained with hematoxylin–eosin and picrofuchsin by Van Gieson for a general assessment of the condition of the examined tissues. Histological micropreparations were studied with a ZEISS Primo Star trinocular microscope (ZEISS Microscopy, Jena, Germany) under 100-, 400- and 1000-times magnification. Microphotographs were taken with a ZEISS Axiocam ERc 5 s (Carl ZEISS Microscopy, Jena, Germany). All the features were examined in accordance with the international standards, WHO recommendations and recognized research methods [50 ].