Immunohistochemical Analysis of TGF-β1 and Smad2/3

We used 5 µm eye sections for the immunohistochemistry after paraffin imbibition, deparaffinization, and rehydration. The primary antibodies used were rabbit polyclonal TGF-β1 (sc-146; Santa Cruz Biotechnology; Dallas, TX, USA) and Smad2/3 (sc-133098; Santa Cruz Biotechnology; Dallas, TX, USA). These were diluted to 1:200 and incubated overnight at 4 °C. Immunoreactions were detected using a Novocastra Peroxidase/DAB kit (Leica Biosystems, Nussloch, Germany). The negative control sections were stained with irrelevant immunoglobulins and analyzed under a bright-field microscope [37 (link)]. The percentage of positive-stained area/total area was quantified using ImageJ software 1.47.

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Trotta M.C., Herman H., Balta C., Rosu M., Ciceu A., Mladin B., Gesualdo C., Lepre C.C., Russo M., Petrillo F., Pieretti G., Simonelli F., Rossi S., D’Amico M, & Hermenean A. (2023). Oral Administration of Vitamin D3 Prevents Corneal Damage in a Knock-Out Mouse Model of Sjögren’s Syndrome. Biomedicines, 11(2), 616.

Publication 2023

TGF-β1 (sc-146; Smad2/3 (sc-133098; Novocastra Peroxidase/DAB kit

Antibodies Immunoglobulins Immunohistochemistry Microscope Paraffin Peroxidase Rabbit Rehydration Smad2 Tgf β1

Corresponding Organization :

Other organizations : University of Campania "Luigi Vanvitelli", Vasile Goldis Western University of Arad

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Variable analysis

independent variables

Primary antibodies used: rabbit polyclonal TGF-β1 (sc-146; Santa Cruz Biotechnology; Dallas, TX, USA) and Smad2/3 (sc-133098; Santa Cruz Biotechnology; Dallas, TX, USA)

dependent variables

Percentage of positive-stained area/total area

control variables

Eye sections of 5 µm thickness
Paraffin imbibition, deparaffinization, and rehydration of the sections
Dilution of primary antibodies to 1:200
Incubation of primary antibodies overnight at 4 °C
Detection of immunoreactions using a Novocastra Peroxidase/DAB kit (Leica Biosystems, Nussloch, Germany)
Negative control sections stained with irrelevant immunoglobulins

controls

Positive control: Not explicitly mentioned
Negative control: Sections stained with irrelevant immunoglobulins

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