The goblets cells were cultivated on slides and treated with RPMI media or diluted eye drops 1:7 (v/v) for 30 min at 37 °C 5% CO. With the use of paraformaldehyde 4% (v/v), the slides were fixated and stored at 4 °C. The cell membranes of the goblet cells were permeated using 0.1% v/v Triton X-100 in PBS, and by using 3% (w/v) bovine serum albumin in PBS, unspecific binding was blocked. The cells were treated with the primary antibodies Cytokeratin-7 (anti-cytokeratin7, 1:500 v/v) and monoclonal anti-human gastric mucin (anti-mucin, 1:200 v/v), diluted in 0.25% bovine serum albumin/0.1% saponin in PBS and washed with PBS thereafter. The fluorescent secondary antibodies, Alexa488 (anti-rabbit, 1:500 v/v) and Texas red (anti-mouse, 1:200 v/v), both diluted in 0.25% bovine serum albumin/0.1% saponin in PBS, were added. Then, 0.3 µM of DAPI in PBS stained the nuclei of the goblet cells. Imaging was performed using an Axioskop 2 (Zeiss; Göttingen, Germany) with an Axio Cam MRm camera (Zeiss; Göttingen, Germany) and an HXP 120 lighting unit (Zeiss; Göttingen, Germany). Image scaling and the merging of pictures were conducted using ImageJ 1.52q (Wayne Rasband, National Institute of Health, Bethesda, MD, USA).
Hxp 120 lighting unit
Manufactured by Zeiss
Sourced in Germany
The HXP 120 is a lighting unit designed for use in microscopy applications. It provides high-intensity, uniform illumination across a wide range of wavelengths. The core function of the HXP 120 is to serve as a light source for various microscopy techniques.
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Lab products found in correlation
2 protocols using hxp 120 lighting unit
1
Immunofluorescence Staining of Goblet Cells
2
Immunofluorescence Staining for Goblet Cell Markers
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GC cultures cultivated on coverslips were incubated for 30 min with Xalatan, latanoprost Pfizer or Monoprost diluted 1:7 (v/v) in culture medium. GCs were incubated with culture medium as negative control and 10−3.5 M histamine as positive control. Cultures were fixed using 4% (v/v) paraformaldehyde and stored at 4°C until immunostaining. The cell membrane was permeated using 0.1% (v/v) Triton X-100 in PBS and non-specific binding was blocked using 3% (w/v) bovine serum albumin (ab181831; Sigma-Aldrich) in PBS. Coverslips were incubated with antibodies to specific markers for cytokeratin-7 (anti-cytokeratin7, 1:500 v/v)(ab181831; Abcam, Cambridge, England) and mucin5AC (anti-mucin, 1:200 v/v)(M5293; Sigma-Aldrich), washed with PBS and incubated with fluorescent secondary antibodies Alexa488 (anti-rabbit, 1:500 v/v)(A11034; Gibco, Life Technologies) and Texas red (anti-mouse, 1:200 v/v)(T862; Gibco, Life Technologies). Nuclei were stained using DAPI (0.3 µM) (D3571; Invitrogen, Massachusetts, USA). A minimum of cultures from three different donors were analysed. Imaging was performed using Axioskop 2 (Zeiss; Göttingen, Germany) with an AxioCam MRm camera (Zeiss; Göttingen, Germany) and HXP 120 lighting unit (Zeiss; Göttingen, Germany). Fiji ImageJ 1.49 was applied for picture optimising and merging.
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