The characteristics of the sori and teliospores were examined on infected plants. Pictures of sori were taken using a Nikon D5100 camera. Teliospore characteristics were studied using a compound microscope (LM; BX60F; Olympus optical Co. Ltd., Japan) equipped with ProgRes C5 camera (JENOPTIK, Germany) and CapturePro software; and a scanning electron microscope (SEM; Hitachi SU6600) at 5.0 kV at the Clemson University Electron Microscopy Facility. For LM, teliospores were mounted in lactic acid (85–90 %, VWR, International, LLC) (Savchenko et al. 2014 (link)) and examined at 1 000 × magnification. The diameters of 30 teliospores, oriented in plane view so that they appeared globose, were measured from each sample collection. The colors of the sori and the teliospores were described according to Rayner (1970) . For SEM examination, teliospores were dusted on double-sided adhesive carbon tape, mounted on aluminum stubs, and sputter-coated with platinum using a Cressington sputter-coater (ca. 30 nm in 6 min).
Bx60f
Manufactured by Olympus
Sourced in Japan, Germany
The BX60F is a versatile laboratory microscope designed for a range of applications. It features a durable construction and provides clear, high-quality images. The BX60F is suitable for various scientific and research purposes.
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6 protocols using bx60f
1
Characterizing Fungal Spore Morphology
2
Quantifying Cellular Proliferation via Ki67
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The proliferation of the cells after 48 h of treatment was determined after immunostaining with anti-Ki67. After 48 h of incubation, the cells were fixed for 30 min in 4% paraformaldehyde (in PBS) and permeabilized for 10 min in PBS containing 0.2% Triton X-100 and 2% bovine serum albumin (BSA) at room temperature. Unspecific binding sites were blocked by incubation with 1% BSA in PBS for 15 min before cells were incubated overnight at 4 °C with the specific anti-Ki67 primary antibody. After washing with PBS, cells were incubated with secondary Alexa Fluor555-conjugated anti-rabbit antibody for 1 h at room temperature. Subsequently, the cells were washed and incubated for 15 min at 37 °C with 3.7% bisBenzimide solution to stain the nuclei and finally sealed with mounting media. Images were captured by fluorescence microscopy (BX60F; OlympusOptical, Tokyo, Japan) and quantified at ImageJ Fiji Software.
3
Histological Analysis of Pancreatic Islets
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For the preparation of histological sections, dorsal skinfold chamber tissue and pancreatic tissue were fixed for 24 h in 4% formalin. In addition, isolated islets were incubated for 45 min at 37 °C in 100 μL HepatoQuick®, 50 μL human citrate plasma and 10 μL 10% CaCl2 solution. The resulting clot was also fixed for 24 h in 4% formalin. The formalin-fixed specimens were embedded in paraffin and 3-μm-thick sections were cut.
The sections were stained with antibodies against insulin (1:300), glucagon (1:300), somatostatin (1:300) and CD31 (1:300) and visualized by their corresponding secondary antibodies. Cell nuclei were stained with Hoechst 33,342. The sections were analyzed by means of fluorescence microscopy (BX60F, Olympus). The number of positively stained cells was determined by FIJI software (NIH) and is given in percentage (%) of all islet cells.
The sections were stained with antibodies against insulin (1:300), glucagon (1:300), somatostatin (1:300) and CD31 (1:300) and visualized by their corresponding secondary antibodies. Cell nuclei were stained with Hoechst 33,342. The sections were analyzed by means of fluorescence microscopy (BX60F, Olympus). The number of positively stained cells was determined by FIJI software (NIH) and is given in percentage (%) of all islet cells.
4
Histological Analysis of Islet-Containing Samples
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For the preparation of histological sections, specimens of islet-containing dorsal skinfold chamber preparations were fixed for 24 h in 4% formalin. In addition, freshly isolated islets were incubated for 45 min at 37 °C in 100 µL HepatoQuick®, 50 µL human citrate plasma and 10 µL 10% CaCl2 solution. The resulting clot was also fixed for 24 h in 4% formalin. The formalin-fixed specimens were then embedded in paraffin and 3-μm-thick sections were cut.
The sections were stained with hematoxylin and eosin (HE) according to standard procedures. For the immunohistochemical analysis, the sections were stained with an anti-insulin, anti-GFP, anti-CD31 and anti-MPO antibody, which were detected by their corresponding secondary antibodies. Cell nuclei were stained with Hoechst 33,342. The sections were analyzed by means of fluorescence microscopy (BX60F; Olympus, Hamburg, Germany). The numbers of insulin-positive, CD31- and GFP-positive islet cells were counted using ImageJ software (NIH, Bethesda, MD, USA) and given in % of all islet cells.
The sections were stained with hematoxylin and eosin (HE) according to standard procedures. For the immunohistochemical analysis, the sections were stained with an anti-insulin, anti-GFP, anti-CD31 and anti-MPO antibody, which were detected by their corresponding secondary antibodies. Cell nuclei were stained with Hoechst 33,342. The sections were analyzed by means of fluorescence microscopy (BX60F; Olympus, Hamburg, Germany). The numbers of insulin-positive, CD31- and GFP-positive islet cells were counted using ImageJ software (NIH, Bethesda, MD, USA) and given in % of all islet cells.
5
Visualizing Anti-CTLA4 Antibody Distribution in Emulsions
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Emulsions were analyzed by optical microscopy (BX60F, Olympus, Japan) at 100× magnification. To evaluate the location of anti-CTLA4 antibodies within the emulsions, we used confocal microscopy (META LSM 510 confocal microscope; Zeiss, Germany). Ipilimumab was conjugated prior with Alexa Fluor 647 (AF647), then labeled antibody was further diluted 100 times with non-conjugated ipilimumab (1% AF647-conjugated ipilimumab). To label the NPs, we first linked covalently PLGA with rhodamine. PLGA–rhodamine was added during the formulation of PLGA NPs to form PLGA NPs (0.1% rhodamine).
6
Quantifying Leukocyte Transmigration and Endothelial ICAM-1 Expression
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Formalin-fixed specimens of the dorsal skinfold chamber preparations were embedded in paraffin. For the analysis of transmigrated leukocytes, 2-mm-thick sections were cut and stained with a polyclonal rabbit antibody against the neutrophil granulocyte marker MPO and a polyclonal rabbit antibody ICAM-1 followed by a biotinylated secondary goat anti-rabbit IgG antibody. Sections solely incubated with the secondary antibody were used as negative controls. The sections were counterstained with Mayer's hemalaun solution and examined by light microscopy (BX60F, Olympus, Hamburg). MPOpositive cells were quantitatively assessed in 10 high power fields (HPFs) per section.
To determine the endothelial expression of ICAM-1 in postcapillary and collecting venules of the dorsal skinfold chamber, we used a semiquantitative score. Values were assigned as follows: 0 ¼ no detectable expression; 1 ¼ mild expression; 2 ¼ moderate expression; and 3 ¼ high expression. This scoring was assessed in 8-10 HPF corresponding to w30 vessels per section.
To determine the endothelial expression of ICAM-1 in postcapillary and collecting venules of the dorsal skinfold chamber, we used a semiquantitative score. Values were assigned as follows: 0 ¼ no detectable expression; 1 ¼ mild expression; 2 ¼ moderate expression; and 3 ¼ high expression. This scoring was assessed in 8-10 HPF corresponding to w30 vessels per section.
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