Human donor eyes were obtained formalin fixed within 6 hours of death from regional eye banks, and were paraffin embedded and sectioned (two 5-μm sagittal sections per slide; n = 8 donors). Sections were deparaffinized, rehydrated, and processed for citrate/heat antigen retrieval, 15 minutes in 100°C citrate buffer (pH 6.0) followed by 15 minutes in room temperature citrate buffer (pH 6.0). Nonspecific staining was blocked by incubation for 15 minutes with 0.05 M glycine/PBS followed by 30 minutes with 5% normal goat serum/PBS. Sections were immunolabeled overnight at 4°C with rabbit anti-TLR4 antibody (1:1000) (Abcam, Cambridge, United Kingdom), washed, and incubated for an hour with secondary antibody. Secondary antibody used was donkey anti-rabbit Alexa Fluor 488 (1:500). Slides were mounted and images acquired using a Nikon Eclipse Ti inverted fluorescence microscope (Nikon, Inc.) equipped with the Cri Nuance FX Camera System (Perkin-Elmer). All images were taken at ×400 magnification; scale bar represents 50 μm.
Cri nuance fx camera system
Manufactured by PerkinElmer
Sourced in United States
The Cri Nuance FX Camera System is a high-performance multispectral imaging device designed for advanced life science applications. It captures and analyzes spectral data from biological samples, enabling researchers to study and visualize complex molecular interactions.
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3 protocols using cri nuance fx camera system
1
Immunolabeling of TLR4 in Human Donor Eyes
2
Fibronectin Immunofluorescence in Murine Eyes
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After completion of the IOP time course, mouse eyes were enucleated and fixed in 4% PFA overnight. Eyes were embedded in paraffin, cut into 5-μm sections, and transferred to glass slides. Deparaffinization was performed by washing two times with xylene, 100% ethanol, 95% ethanol, and 50% ethanol for 2 minutes each. Slides were then soaked in PBS for 5 minutes. Tissues were blocked using Superblock Blocking Buffer in PBS (Thermo Fisher Scientific) for 30 to 60 minutes. Rabbit anti-fibronectin (EMD Millipore) 1:1000 dilution was used to label FN, followed by Alexa Fluor–labeled anti-rabbit Ig (Life Technologies) 1:1000 dilution. Prolong Gold mounting medium containing DAPI (Invitrogen-Molecular Probes) was used to mount the slides and imaged using fluorescent microscope Nikon ECLIPSE Ti-U (Nikon Instruments, Inc., Melville, NY, USA) equipped with a CRi Nuance FX Camera System (Perkin-Elmer, Waltham, MA, USA). All images were taken at ×400 magnification; scale bar represents 50 μm.
3
Visualization and Quantification of Cytoskeletal Actin Networks
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CLANs were visualized using the Nikon Eclipse Ti inverted fluorescence microscope (Nikon, Inc., Melville, NY, USA) with 600× magnification. Cytoskeletal images were taken using the Nikon Eclipse Ti inverted fluorescence microscope equipped with the Cri Nuance FX Camera System (Perkin-Elmer, Inc., Waltham, MA, USA).
CLANs were defined as F-actin–containing cytoskeletal structures with at least one triangulated actin arrangement consisting of actin spokes and at least three identifiable hubs.46 (link) Representative images of CLANs are shown inFigures 1 A–1 C. Each coverslip was assessed at 10 locations (Fig. 1 D) with approximately 100 to 150 cells per coverslip. Six to 12 coverslips were evaluated per treatment group.
All CLAN counting was done in a masked manner. CLAN-positive cells (CPCs) were defined as any cell containing at least one CLAN or multiple CLANs. The formation of CLANs was compared using the percentage of CPCs, which is calculated by dividing the number of CPCs by the number of DAPI-positive cells.
CLANs were defined as F-actin–containing cytoskeletal structures with at least one triangulated actin arrangement consisting of actin spokes and at least three identifiable hubs.46 (link) Representative images of CLANs are shown in
All CLAN counting was done in a masked manner. CLAN-positive cells (CPCs) were defined as any cell containing at least one CLAN or multiple CLANs. The formation of CLANs was compared using the percentage of CPCs, which is calculated by dividing the number of CPCs by the number of DAPI-positive cells.
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