After treatments, RHEs were lysed with specific lysis buffer (T-PER, Thermo Scientific, #78510, Waltham, MA, USA) to evaluate filaggrin, loricrin and claudin-1 expressions. Protein concentration was determined by a BCA assay, and then lysates were kept frozen at −80 °C until use. All the samples were adjusted to the same protein concentration, and an equal quantity of proteins was loaded in each capillary. Target proteins were identified by a capillary electrophoresis-based protein analysis system (Sally Sue; ProteinSimple, San Jose, CA, USA) using primary antibodies (anti-filaggrin, clone AKH1, Santa Cruz Biotechnologies #sc66192, Dallas, TX, USA; anti-loricrin, Abcam #ab176322; anti-claudin-1, Life technologies #71-7800, Carlsbad, CA, USA), and they were immunoprobed using a horseradish peroxidase-conjugated secondary antibody and chemiluminescent substrate. The capillaries containing a proprietary UV-activated chemical-linked coating were obtained from ProteinSimple. All samples and reagents were prepared according to the recommended manufacturer’s instructions. The resulting chemiluminescent signal was detected and quantified using Compass Software version 2.7.1 (ProteinSimple, Minneapolis, MN, USA) followed by a statistical analysis.
Anti loricrin
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-loricrin is a laboratory product that can be used for research purposes. It is a protein that binds to the loricrin protein, which is a structural component of the skin. The function of anti-loricrin is to facilitate the study of the loricrin protein and its role in skin biology.
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Lab products found in correlation
Anti filaggrin, by Abcam (3 mentions)
Lsm 700, by Zeiss (2 mentions)
T per, by Thermo Fisher Scientific (1 mentions)
Anti claudin 1, by Thermo Fisher Scientific (1 mentions)
Gastrin, by Santa Cruz Biotechnology (1 mentions)
Anti gfp, by Abcam (1 mentions)
Anti cxcr2, by Abcam (1 mentions)
Anti p63, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 donkey anti mouse, by Thermo Fisher Scientific (1 mentions)
Anti keratin 14, by Abcam (1 mentions)
Dm750, by Leica (1 mentions)
Anti trpa1 gtx54765, by GeneTex (1 mentions)
Permount sp15 100, by Thermo Fisher Scientific (1 mentions)
Ultravision quanto detection system tl 060 qhd, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated goat anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti cd3, by Abcam (1 mentions)
Anti ki67 antibody, by Abcam (1 mentions)
Anti cytokeratin 5, by Abcam (1 mentions)
Anti involucrin, by Santa Cruz Biotechnology (1 mentions)
Vectashield mounting medium with 4 6 diamidino 2 phenylindole, by Vector Laboratories (1 mentions)
9 protocols using anti loricrin
1
Protein Expression Analysis in RHEs
2
Comprehensive IHC Staining Protocol
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For IHC, tissues were harvested, fixed in 10% neutralized Formalin for 2 days and then stored in 70% ethanol until further processing. H&E staining, PAS staining and IHC on paraffin-sections were performed following standard protocols. The following antibodies were used for IHC: anti-GFP (Abcam, 6673, 1:200; Clontech, JL-8, 1:100); Ki67 (Cell signaling, 12202, 1:200); Proton-pump (MBL, D032-3H, 1:100); Gastrin (Santa Cruz, sc-783, 1:200); anti-p63 (Santa Cruz, sc-56188, 1:200); anti-CK13 (Abcam, 92551, 1:1000); anti-Loricrin (Abcam, 24722, 1:1000); anti-CXCL7 (Bioss Inc., A-21235, 1:200); anti-CXCR2 (Abcam, 14935, 1:200).
3
Immunofluorescence Staining of Keratins
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All chemicals were purchased from Sigma-Aldrich (Saint Quentin Falavier, France). The antibodies used in the study were Anti-keratin 14 (Abcam, Cambridge, MA, USA, mouse monoclonal), Anti-keratin 16 (Abcam, Cambridge, MA, USA, mouse monoclonal), Anti-loricrin (Abcam, Cambridge, MA, USA) mouse monoclonal, Alexa Fluor 488 donkey anti-mouse (Thermofisher scientific, Montigny le Bretonneux, France).
4
Immunohistochemical Analysis of Skin Samples
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Each tissue slide was prepared as described previously [69 (link)]. Staining was carried out via H&E and toluidine blue, followed by immunohistochemical staining with anti-filaggrin (Abcam, Cambridge, UK), anti-loricrin (Abcam), and anti-TRPA1 (GTX54765; GeneTex, Irvine, CA, USA) antibodies. Staining was performed using an Ultravision Quanto Detection System (TL-060-QHD; Thermo Fisher Scientific, Waltham, MA, USA). After staining, tissues were dehydrated and sealed in Permount (SP15-100; Thermo Fisher Scientific), and observed via optical microscopy (DM750, Leica, Wetzlar, Germany). For fluorescence images, the sections were incubated with primary antibody with anti-TRPA1 (GTX54765; GeneTex, Irvine, CA, USA) antibody at 4 °C for 16 h, followed by incubation with secondary antibodies against FITC-conjugated goat-anti-rabbit IgG (1:1000, sc-2012, Santa Cruz Biotechnology) at 21 °C for 1 h. Immunostained tissues were mounted with a medium containing Fluoroshield™ with DAPI (ImmunoBioScience, Mukilteo, WA, USA). Fluorescence images were acquired by using a confocal microscope (LSM700; Zeiss, Jena, Germany).
5
Immunohistochemical Analysis of Mouse Skin
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The mouse back skin was fixed in formalin and embedded in paraffin. Partial sections were stained with hematoxylin-eosin (H&E) for pathological observation by microscopy. Partial sections were stained with anti-Rabbit CD3 (1:100, Abcam, United States) and anti-Loricrin (1:200, Abcam, United States), and Diaminobezidin (DAB) (Zhongshan Golden Bridge Biotechnology, China) was used for color development. For immunofluorescence, Ki-67 (proliferation) level was evaluated in skin sections using anti-Ki67 antibody (1:200, Abcam), following the manufacturer’s instructions. For CD3+, Ki-67+, and Loricrin+ cells, three areas in three sections of each sample were selected randomly, and the number of positive cells was calculated.
6
Immunohistochemical Analysis of Epidermal Markers
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All chemical reagents were obtained from Sigma-Aldrich. The antibodies were as follows: anti-loricrin (Abcam, ab24722), working dilution for immunohistochemistry (IHC) 1/1000; anti-involucrin (Santa Cruz, sc-15230), working dilution for IHC 1/1000; anti-cytokeratin 5 (Abcam, ab24647), working dilution for IHC 1/1000; antidesmoglein 1 (Santa Cruz, sc-20114), working dilution for IHC 1/300; anti-desmocollin 1 (Santa Cruz, sc18115), working dilution for IHC 1/250; anti-corneodesmosin (CusAb, CSBPA0051241A01HU), working dilution for IHC 1/500.
7
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.
8
Immune Response Tissue Analysis
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For confirming immune response from tissues, immunofluorescence staining was conducted following standard protocols. Cross-sectioned slides were blocked with 4% bovine serum albumin solution (Sigma-Aldrich, USA), incubated with primary antibodies overnight, and followed standard washing protocol. Alex Fluor 594 anti-cluster of differentiation 68 (CD68 at 1:100; Santa Cruz Biotechnology), Alex Fluor 488 anti-cluster of differentiation 206 (CD206 at 1:100; Santa Cruz Biotechnology), anti-caspase-3 (1:100; Santa Cruz Biotechnology), and anti-Loricrin (Loricrin at 1:100; Abcam) were used to investigate the distribution of macrophages, apoptosis, and epithelium in in vivo experiments. The CD68 + , CD 206 + , and caspase-3+ cells ratio was calculated as the percentage of the total cell area consisting of CD68- or caspase-3-area.
9
Quantification of Skin Barrier Proteins
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Cells and dorsal tissues were collected and lysed in PRO-PREP (iNtRON, Seongnam, Korea) containing a protease inhibitor cocktail (Complete™; Roche, Mannheim, Germany). Amounts of protein in the lysates were quantitated using a BCA kit (Thermo Fisher Scientific). Following quantitation, equal amounts of protein were resolved on 10% SDS-PAGE gels and electrotransferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). After blocking with 5% skim milk in Tris-buffered saline containing 0.5% Tween-20 (TBST), membranes were probed with anti-filaggrin, anti-involucrin, anti-loricrin from Abcam (Cambridge, UK); anti-alix, anti-flotillin, anti-CD9, anti-phospho-STAT1, anti-STAT1, anti-phospho-STAT1, anti-STAT1, anti-phospho-STAT3, anti-STAT3 from Cell Singling Technology (Danvers, MA, USA); and anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies at 4 °C overnight. After washing, membranes were incubated with HRP-conjugated anti-mouse (Vector Labs Inc., Burlingame, CA, USA) or anti-rabbit (Vector Labs Inc.) secondary antibodies. Immunoreactive signals were detected using enhanced chemiluminescence (ECL) reagents (EzWestLumi plus; ATTO, Tokyo, Japan).
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