Paraffin-embedded 4 μm sections of skin biopsies were deparaffinized in xylene and rehydrated in decreasing alcohol gradients. Antigen retrieval was achieved using heat-based epitope retrieval with sodium citrate buffer (Thermo Fisher Scientific, USA) (pH 6.0) at a sub-boiling temperature in a 500 W microwave for 10 min. After cooling, sections were washed in PBS, blocked with 5% BSA in PBS, and incubated with primary antibodies for 4HNE (dil. 1:500) (AB5605, Millipore), NFκB (dil. 1:500) (8242, Cell Signaling), AhR (dil. 1:500) (83,200, Cell Signaling), involucrin (dil. 1:50) (sc-21748, Santa Cruz), or filaggrin (dil. 1:50) (sc-66192) in 2% BSA in PBS. Sections were then washed in PBS and incubated with fluorochrome-conjugated secondary antibodies (dil. 1:1000) (Alexa Fluor 568 A11004 or Alexa Fluor 488 A11055) in 2% BSA in PBS at RT, and then washed with PBS. Nuclei were stained with DAPI (1,874,814, Invitrogen) in PBS, and sections were then washed with PBS. Sections were mounted using PermaFluor mounting media (ThermoFisher Scientific) and imaged on a Zeiss LSM10 microscope. Images were quantified using ImageJ.
Sc 21748
Manufactured by Santa Cruz Biotechnology
Sc-21748 is a lab equipment product offered by Santa Cruz Biotechnology. It is designed to perform a core function related to biotechnology research and analysis, but a detailed description cannot be provided while maintaining an unbiased and factual approach. Further information is not available.
Automatically generated - may contain errors
Lab products found in correlation
Sc 66192, by Santa Cruz Biotechnology (2 mentions)
Ab5605, by Merck Group (1 mentions)
Sodium citrate buffer, by Thermo Fisher Scientific (1 mentions)
Permafluor mounting media, by Thermo Fisher Scientific (1 mentions)
Lsm10 microscope, by Zeiss (1 mentions)
Sc 47778, by Santa Cruz Biotechnology (1 mentions)
Hyperfilm, by GE Healthcare (1 mentions)
2 protocols using sc 21748
1
Immunohistochemical Analysis of Skin Biopsies
2
Keratinocyte Differentiation Assay
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Differentiation of primary keratinocytes: Normal and SCC7 primary keratinocytes were grown in 6 cm dishes in 50% and full confluence (96 hours growth) or under high Ca2+ conditions (1.5mM CaCl2) in CnT07 keratinocyte medium (CELLnTEC, Bern, Switzerland). Cells were grown in the presence of Ca2+ for 24h, 48h and 96h. As positive control cells grown in a low Ca2+- medium for 96 hours was used where cells have reached full confluency and passage-driven differentiation. Cells were lysed in RIPA buffer (conventional protocol). The lysates were collected and subjected to SDS-PAGE, followed by immunoblotting using specific antibodies against human TLR4 (H-80, sc10741, 1:100 dilution), involucrin (sc-21748, 1:100 dilution), filaggrin (sc-66192, 1:100 dilution) and actin (sc-47778, 1:200 dilution), all from Santa Cruz Biotechnology. Incubation with primary antibodies was performed at 4°C, overnight. Incubation with secondary rabbit anti-mouse IgG H&L HRP (ab6728) was performed at room temperature for 1 hour. Proteins were detected by ECL on Hyperfilm (Amersham, GE Healthcare).
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