In 8-well chamber slides, KCs (1 × 104 cells/well) were seeded and treated with the positive control agent (1.8 mM calcium) or 0.05% H.ECM™ liposome when they reached over 80% confluence and incubated for 24 h. For immunofluorescence, the 8-well chamber slide containing treated KC cells and human skin cryosections (6 μm-thick) were fixed at room temperature (20–25 °C) with 4% (w/v) paraformaldehyde (Cell Signaling Technology, Danvers, MA, USA) for 15 min. Post-fixation, the chamber or cryosections were washed with phosphate-buffered saline (PBS)-T (PBS containing Triton X-100; DAEJUNG, Busan, KR) three times, followed by incubation with anti-filaggrin (ab81468; Abcam) or nti-loricrin (ab85679; Abcam) antibodies overnight at 4 °C. After washing three times with PBS-T, samples were incubated with the FITC-labeled secondary fluorescent antibody (Goat pAb to Rb IgG-FITC, ab6717; Abcam) for 2 h at room temperature to bind primary antibodies. Finally, the cells and tissue specimen were fixed using the fixation solution (VECTASHIELD® mounting medium with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories Inc., Burlingame, CA, USA). All fluorescence images were acquired using a laser-scanning microscope (LSM 700, Carl Zeiss, Jena, Germany), and fluorescence intensity was measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Paraformaldehyde (pfa)
Manufactured by Cell Signaling Technology
Sourced in United States
Paraformaldehyde is a powdered, polymerized form of formaldehyde. It is a commonly used fixative for preserving and stabilizing biological samples prior to analysis or imaging.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700, by Zeiss (4 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Ab85679, by Abcam (1 mentions)
Ab81468, by Abcam (1 mentions)
Vectashield mounting medium with 4 6 diamidino 2 phenylindole dapi, by Vector Laboratories (1 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (1 mentions)
Anti filaggrin, by Abcam (1 mentions)
Anti loricrin, by Abcam (1 mentions)
Vectashield mounting medium with 4 6 diamidino 2 phenylindole, by Vector Laboratories (1 mentions)
Triton x 100, by Cell Signaling Technology (1 mentions)
Freezing microtome, by Leica (1 mentions)
Th primary antibody, by Abcam (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (1 mentions)
Synergy h1 microplate reader, by Agilent Technologies (1 mentions)
Xds 2, by Optika (1 mentions)
Alizarin red s, by Merck Group (1 mentions)
5 protocols using paraformaldehyde (pfa)
1
Immunofluorescence Analysis of Keratinocyte Differentiation
2
Immunofluorescence Staining of Mouse Skin Cryosections
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For immunofluorescence staining, slide-mounted mouse skin cryosections (6 µm thick) were fixed using 4% paraformaldehyde (Cell Signaling Technology, Danvers, MA, USA) for 15 min at room temperature (RT). Slides were washed three times with phosphate-buffered saline (PBS)-T (PBS containing Triton X-100; DAEJUNG, Busan, Korea). Slides were incubated with primary antibodies overnight at 4 °C. After being washed three times, slides were incubated with a secondary antibody (Goat pAb to Rb IgG-FITC; ab6717; Abcam) for 1 h at RT. Next, sections were rinsed three times with PBS and DAPI (VECTASHIELD® mounting medium with DAPI, Vector Laboratories Inc., Burlingame, CA, USA) was applied for nuclear staining. Slides were scanned using a Zeiss microscope (LSM 700; Carl Zeiss, Jena, Germany) for image acquisition, and the fluorescence intensity was measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
3
Quantifying Keratinocyte Response to UVB and ZAG
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KC cells (2.5 × 104 cells/well) were seeded in 4‐well chamber slides and incubated for 24 h before treatment with UVB (10 J/cm2) or UVB (10 J/cm2) + ZAG peptide (2.0 µg/mL). Cells were then fixed with 4% (w/v) paraformaldehyde (Cell Signaling Technology, Danvers, MA, USA) for 15 min at room temperature, followed by three washes with PBS. The fixed cells were incubated overnight with anti‐loricrin (Abcam Cambridge, UK) or anti‐filaggrin (Abcam) antibodies at 4°C. Thereafter, the slides were washed three times and incubated with FITC‐labeled secondary fluorescent antibodies (Goat pAb to Rb IgG‐FITC; Abcam) for 2 h at room temperature. Finally, the slides were fixed with VECTASHIELD mounting medium with 4′,6‐diamidino‐2‐phenylindole (Vector Laboratories Inc., Burlingame, CA, USA). Fluorescence images were obtained using a laser‐scanning microscope (LSM 700, Carl Zeiss, Jena, Germany) and analyzed using ImageJ software.
4
AAV-mediated miRNA Delivery in MPTP Mouse Model
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6 male B6 wild-type mice were raised under specific pathogen-free environment for 1 year. AAVs carried miRNA (Obio Technology, China) were injected to the substantia nigra pars compacta (SNc) of the mice brain through stereotaxic surgery. The coordinate axis parameter of SNc is ±1.25 mm in M/L, −3.16 mm in A/P, −4.25 mm in D/V. After 3 weeks, the MPTP (Sigma, United States) was administrated in these mice with injection every 2 h for four doses in total over an 8 h period (20 mg/kg per dose × 4). One-day interval later, these mice were sacrificed and their brains were removed. The brain tissues were fixed with 10% paraformaldehyde (Cell Signaling Technology, United States) for 1 day at room temperature, followed by dehydration in 30% sucrose for 2 days at 4°C and embedding with O.C.T.Compound (Solarbio, United States) at −80°C. The brain tissues were cut to 20 μm thick slices with the freezing microtome (Leica, Germany). The slices were blocked with 5% BSA powder (Proliant, United States) dissolved in PBS containing 0.3% Triton X-100 (Cell Signaling Technology, United States) for 1 h followed by incubation of rabbit tyrosine hydroxylase (TH) primary antibody (Abcam, United States, 1:200) overnight at 4°C and then with Fluor 594 (red) secondary antibody (ThermoFisher, United States) at 37°C for 30 min. Images were taken by Zeiss LSM 880 confocal microscope.
5
Quantification of Osteogenic Mineralization
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Control and differentiated cells were grown in 24 well plates. On a specific day (5th, 11th, 15th, 20th, 24th) after osteogenic medium application, cells were fixed by 4% paraformaldehyde (Cell Signaling Technology, Danvers, MA, USA) for 30 min at RT. After DPBS (Gibco, Paisley, UK) washing, formed Ca2+ deposits were stained by 2% Alizarin Red S (Merck Sigma-Aldrich, Darmstadt, Germany) for 3 min at RT. Next, the dye was aspired and cells were carefully washed three times by ultrapure water to remove the remaining stain. Finally, cells were kept in ultrapure water. Color intensity was monitored and images were captured by the inverted microscope XDS-2 (Optika). For quantification of total stained mineralized tissue, Alizarin Red S was eluted by destain solution (10% acetic acid, 20% methanol) for 15 min at RT [111 (link)]. The eluates were centrifuged (10,000× g) for 10 min at RT to remove mineral precipitates and cell debris. Then, the supernatant was transferred into 96 well plates in duplicates. The absorbance was measured at 405 nm using Synergy H1 Microplate Reader (BioTek, Winooski, VT, USA). The amount of mineral deposits is presented as optical density normalized to the total amount of proteins (mg), which were determined by BCA assay (Thermo Fisher Scientific).
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