General morphological structure of different epidermal layers in tissues were analyzed using hematoxylin and eosin (H&E) staining. In addition, the NF-κB pathway was evaluated by immunofluorescence for p65 subunit. As previously described, following O3 exposure, tissues (N = 3 per condition) were placed at 37 °C in the 5% CO2 incubator overnight. After 24 h, tissues were fixed by immersion in 10% neutral-buffered formalin (NBF) at RT and then paraffin embedded. Sections (4 μm) were deparaffinized in xylene and rehydrated in alcohol gradients, before H&E staining or immunofluorescence analysis. For immunofluorescence, after a heat-mediated antigen retrieval step in pH 6.0 citrate buffer, tissues were blocked in 5% normal goat serum for 1 h and, then, incubated with NF-κB p65 antibody overnight at 4 °C. Slides were then incubated with a secondary fluorescent goat anti-rabbit Alexa Fluor 488 antibody for 1 h at RT in the dark. The slides were mounted with ProLong Gold Antifade Mountant with 4′, 6-diamidino-2-phenylindole (DAPI) for nuclei staining. Negative control sections were processed by omitting primary antibody. Images were acquired using a Zeiss Z1 AxioObserver LSM10 confocal microscope equipped at 40 × magnification.
Z1 axioobserver lsm10 confocal microscope
Manufactured by Zeiss
Sourced in United States
The Z1 AxioObserver LSM10 is a confocal microscope designed for high-resolution imaging. It features a laser scanning system and a motorized stage to capture detailed images of samples.
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2 protocols using z1 axioobserver lsm10 confocal microscope
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Epidermal Morphology and NF-κB Pathway
2
Immunofluorescence Analysis of Inflammasome Proteins
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HaCaT cells or 1° KC were grown on 10 mm2 coverslips at a density of 70’000 cells/coverslip in 24 wells plates. Cells were fixed in 4% paraformaldehyde (PFA) in PBS for 10 min at RT, permeabilized with 0.25% of Triton X-100 in PBS for 10 min and then blocked in PBS containing 1% BSA at room temperature for 30 min as previously described [44 (link)]. Coverslips were incubated with primary antibody ASC (Cat. NBP1-78977 Novus Biological, Littleton, CO, USA) 1:100, NLRP1 (sc-166368 Santa Cruz Biotechnology Inc., Dallas, TX, USA) 1:50, 4HNE (ab46545, Abcam, Cambridge, UK) 1:400, DPP9 (ab42080, Abcam, Cambridge, UK)1:400, UBR2 (18852-1-AP, Proteintech Group Inc., Rosemont, IL, USA) 1: 150 in 0.25% BSA/PBS overnight at 4°C. The Alexa Fluor Fluorochrome-conjugated secondary antibodies (A11004 Alexa Fluor 568, A11008 Alexa Fluor 488 Invitrogen, ThermoFisher Scientific, USA) were used at a dilution of 1:1000 in PBS-BSA 0.25% for 1 h at RT. Nuclei were stained with DAPI (D1306 Invitrogen ThermoFisher Scientific, Waltham, MA, USA) after removal of secondary antibody. Coverslips were mounted onto glass slides using PermaFluor Aqueous Mounting Medium (TA-006-FM, ThermoFisher Scientific, Waltham, MA, USA), and examined using a Zeiss Z1 AxioObserver LSM10 confocal microscope equipped at 40x and 60x magnification. Images were quantified using ImageJ software.
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