WT spleens were fixed in 4% paraformaldehyde (PFA) in 0.1M sodium phosphate buffer (pH 7.4) overnight. Following transcardial perfusion with 4% PFA, optic nerves/chiasms were dissected and post-fixed in 4% PFA. Slides were deparaffinized in xylene, subjected to antigen retrieval, and incubated overnight with the appropriate antibodies (Supplementary Table S1 ), followed by incubation with biotinylated secondary antibodies (1:200; Vector Laboratories, Burlingame, CA). Signals were amplified with the Vectastain ABC kit (PK4000, Vector Laboratories), developed using the Vectastain DAB kit (SK4100, Vector laboratories), and counterstained with hematoxylin. Specimens were scanned using a NanoZoomer 2.0-HT slide scanner (Hamamatsu Photonics K.K., Japan) with a 20x objective, and the percentage of immunopositive cells calculated by manual counting. Optic nerve sections were deparaffinized, hydrated and stained in toluidine blue (1% toluidine blue in 70% ethanol; pH 2.3) for 3 minutes, washed in distilled water, dehydrated, and mounted. Stained optic nerves were imaged for quantification on a Leica ICC50W microscope using LAS EZ software.
Icc50w microscope
Manufactured by Leica camera
Sourced in United States, Germany
The ICC50W is a high-quality microscope from Leica. It features a binocular optical head with Plan Achromat objectives for sharp, high-contrast images. The microscope is designed for basic routine applications in life science laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Biotinylated secondary antibody, by Vector Laboratories (1 mentions)
Vectastain dab kit, by Vector Laboratories (1 mentions)
Nanozoomer 2.0 ht slide scanner, by Hamamatsu Photonics (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
Fluorescein diacetate fda, by Merck Group (1 mentions)
Dm500 microscope, by Leica camera (1 mentions)
Lacv monoclonal antibody 8c2.2, by Thermo Fisher Scientific (1 mentions)
8 protocols using icc50w microscope
1
Immunohistochemistry and Toluidine Blue Staining of Optic Nerves
2
Murine Blood Cell Profiling
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Thin blood films prepared from murine blood were left to air-dry overnight, fixed for 60 s in methyl alcohol and stained with 10% Giemsa. A Leica ICC50W microscope was used to (i) morphologically profile the blood cells, (ii) count leucocytes, erythrocytes and platelets in the cell monolayer (10 fields) and (iii) record cell irregularities, such as erythrocyte agglutination or platelet aggregation. As the platelets were often aggregated, we were unable to generate accurate total platelet counts. To qualitatively assess the levels of platelet clumping we, therefore, used the following grading system: (i) no aggregation, <1 small colony/field; (ii) mild aggregation, 2–5 colonies/field and/or small colonies only; (iii) moderate aggregation, 2–8 colonies/field and/or small and medium colonies; (iv) marked aggregation, >5 colonies/field and/or medium and large colonies present. Small, medium and large colonies were defined as 2–10 platelets, 11–20 platelets and >20 platelets, respectively.
3
Analyzing Toxoplasma Gondii Parasite Replication
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1×105 ME49wt parasites or ME49Δcdpk3 parasites were used to infect MEF monolayer seeded on coverslips for 1 hours in a 12 well plate. Cells were washed twice with PBS and resuspended in normal culture medium. 24 hours after parasite infection, cells were fixed in 4% paraformaldehyde and stained for Giemsa staining. The number of parasites each parasitophorous vacuole (PV) (i.e., one, two, four, eight) was obtained by counting ≥100 PVs using a LEICA ICC50W microscope at 1000×magnification. The infected cells were processed for immunoblot and qPCR as previously described. All strains were tested 3 times or more independently, each with three internal replicates.
4
Neurite Outgrowth Measurement in Neuroblastoma
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Neuroblastoma SH-SY5Y cells were plated in glass bottom dishes, with 22 mm of glass diameter at a density of 48 × 103 cells per dish in complete medium DMEM-F12 and 10% FBS, for 24 h. Thereafter, both differentiated (see Section 2.6.1 ) and undifferentiated SH-SY5Y cells were treated with the diverse samples at a concentration of 1 µM for each peptide and 2 µg/mL for bwGO for 2 h. Optical bright field images were recorded with a Leica ICC50 W microscope immediately after the treatment (t = 0) and after 2 h of incubation and analyzed to assess the neurite outgrowth, using the NeuronJ (neurite tracing and quantification) plugin from the ImageJ Software (NIH, Bethesda, MD, USA).
5
Cytoplasm and Organelle Visualization
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To visualize surviving cytoplasm, cells were stained with 2 μg ml–1 fluorescein diacetate (FDA) (Sigma-Aldrich, United States) for 5–10 min or glycogen inside cytoplasm was stained with solutions containing 60 mg ml–1 KI and 10 mg ml–1 I2 for 2 min (Thines et al., 2000 (link)). To label endolysosomes and vacuoles, 7-amino-4-chloromethylcoumarin (CMAC, Invitrogen, United States) solution (10 μM final concentration) was added to spore suspensions, and the spores were inoculated on hydrophobic borosilicate glass coverslips and incubated for 24 hpi. Samples were observed under a fluorescence microscope (Nikon Eclipse Ni, Japan) or a Leica icc50 W microscope (Leica, Germany). The experiments were performed three times with three replicates each time, and 110–200 spores or appressoria were counted per replicate.
6
Cryosectioning and Histological Staining
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Frozen sections (5 µm) were cut with a cryostat and placed on slides. After being fixed with 100% acetone for 5 min and dried at room temperature, the slides were exposed to deionized water, hematoxylin (1:4 dilution), bluing reagent, and eosin consecutively, followed by stepwise dehydration, cleaning, and mounting procedures. The slides were visualized under a Leica ICC50W microscope. Cell density was determined using the ImageJ software application with the CellProfiler option for Windows (NIH, 1.53n/2021; github.com/imagej/imagej1).
7
Ovarian Follicle Counting and Corpus Luteum Analysis
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Ovaries (n = 5 per treatment) were embedded in paraffin at the Iowa State University Veterinary Medicine Histopathology Laboratory and sectioned at 5 μM thickness. Every sixth section was mounted on a slide, with two sections per slide. Tissue sections were stained with hematoxylin and eosin, and slides were blinded before counting follicles to remove counter bias. Healthy follicles on every 12th section were counted using a Leica DM 500 microscope equipped with ICC50W microscope. Healthy follicles contained an oocyte nucleus whereas unhealthy follicles were distinguishable by demonstration of pyknosis and intense eosinophilic staining. Follicles were classified as primordial, primary, secondary, pre-antral, and antral following the procedures described previously [41 (link),42 ]. The number of corpora lutea which are oocyte-devoid was averaged across all the sections of each ovary.
8
Immunohistochemistry of LACV in Aedes Mosquitoes
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Ae. aegypti HWE and Ae. albopictus LC females received an LACV-containing bloodmeal (BM-1) (titer: 1.3 × 106 PFUs/mL) followed by a non-infectious bloodmeal (BM-2) at 10-days post-BM-1. Mosquito bodies (without legs and wings) were collected at 24 h post-BM-2 and fixed in 10% neutral-buffered formalin at 4 °C for a minimum of 24 h. Mosquito bodies were then embedded in paraffin and sectioned onto microscope slides for IHC staining. Slides were incubated with LACV monoclonal antibody (8C2.2) (Invitrogen) at a dilution of 1:200 and incubated with a Mouse Envision secondary antibody for 20 min. All IHC staining was performed by the MU Veterinary Medicine Diagnostic Laboratory. Slides were imaged using a Leica ICC50 W microscope equipped with a Wi-Fi-enabled camera.
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