To further establish the neuroblastoma cell cultures as a reliable neuronal model, we differentiated SH-SY5Y and #9 neuroblastoma cells by following a published 18-day protocol (Shipley et al., 2016 (link)). This protocol includes carefully timed supplementation with three separate differentiation medias, which allows for gradual serum-starvation and the addition of retinoic acid, extracellular matrix proteins and neurotrophic factors. Over time, the cells develop neuron-like morphologies, resulting in a representative expansive and networked neuronal culture (Fig. 6G ). Cultures were plated on extracellular matrix coated plates or PDL coated coverslips (Neuvitro, Cat# GG121.5PDL) for the final step of differentiation and subsequent cellular assays and immunohistochemistry. Brightfield, live cell images were obtained with 20x magnification from a Zeiss Primovert inverted phase contrast microscope.
Primovert inverted phase contrast microscope
Manufactured by Zeiss
Sourced in Germany
The Primovert inverted phase contrast microscope is a compact and versatile laboratory equipment designed for various microscopy applications. It features a phase contrast optical system that enhances the contrast of transparent specimens, enabling clear visualization of cellular structures and details. The microscope's inverted configuration allows for convenient sample observation from below, making it suitable for a range of cell culture and live-cell imaging applications.
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3 protocols using primovert inverted phase contrast microscope
1
Neuroblastoma Cell Differentiation Protocol
2
Histochemical Evaluation of Osteoblast ALP
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Extracellular ALP was qualitatively evaluated by histochemical staining at day 4 after induction of osteoblast differentiation. For such purpose, C2C12 cells were washed twice with PBS and subsequently fixed with 4% paraformaldehyde at RT for 5 min. The fixed cells were further washed twice with PBS and stained with a solution containing Fast Blue RR salt (Sigma Aldrich/Merck, Steinheim, Germany) and Naphthol AS-MX phosphates alkaline solution (Sigma Aldrich/Merck, Steinheim, Germany) at RT for 2 h. After discarding the staining solution and washing the plates with PBS, the cells were observed using Primovert inverted phase contrast microscope (ZEISS, Oberkochen, Germany). The resulting blue, insoluble, granular dye deposit indicates sites and the intensity of ALP activity.
3
Sirius Red Staining for Liver Fibrosis
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Sections were equilibrated to room temperature before being incubated in 0.1% Sirius Red (Sigma “Direct Red 80”) for 1 h. The sections were then washed in acidified water twice and dipped in 100% EtOH three times. They were cleared in xylene and mounted with Cytoseal XYL. The sections were viewed on a Zeiss Primo Vert Inverted Phase Contrast microscope and imaged with an attached Axiocam 105 color camera. In ImageJ (NIH), images were converted into 8-bit files and underwent thresholding. Regions of interest were drawn on liver sections, and fibrosis was measured as percent area positive for staining.
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