Immunofluorescent Analysis of Innate Immune Receptors

Deparaffinized sections were treated with a 5% solution of sodium borohydride to eliminate autofluorescence, then a 2.5% solution of bovine serum albumin (BSA) was used, and then slices were incubated overnight with primary antibodies against TLR2, CD14, and α-Tubulin [40 (link)]. After that, the incubation of secondary antibodies was performed. To avoid bleaching, the slices were mounted using Fluoromount™ Aqueous Mounting Medium (Sigma-Aldrich, Taufkirchen Germany, Europe). Experiments were run without the primary antibodies as a negative control (data not shown). In order to confirm that the primary antibodies were immunopositive, rat skin tissues were employed as a positive control (data not shown) [5 (link), 47 (link), 74 (link)].
The sections were analyzed under a Zeiss LSM DUO confocal laser scanning microscope with a META module (Carl Zeiss MicroImaging GmbH, Jena, Germany, Europe) with a helium–neon (543 nm) and argon (458 nm) lasers of different wavelengths [7 ]. To improve pictures Zen 2011 (LSM 700 Zeiss software, Oberkochen, Germany, Europe) was used. Using Adobe Photoshop CC version 2019 (Adobe Systems, San Jose, CA, USA) the digital images were merged to a composite figure. The fluorescence intensity curves were then evaluated using Zen 2011 "Display profile" feature [58 (link)]. Details about antibodies are summarized in Table 2.Table 2

Antibodies data

Antibody	Supplier	Dilution	Animal source
CD14	Santa Cruz Biotechnology, Inc., Dallas, TX, USA	1:200	Mouse
TLR2	Active Motif, La Hulpe, Belgium, Europe	1:125	Rabbit
α-Tubulin	Santa Cruz Biotechnology, Inc., Dallas, TX, USA	1:500	Mouse
Alexa Fluor 488 Donkey anti-Mouse IgG FITC conjugated	Molecular Probes, Invitrogen	1:300	Donkey
Alexa Fluor 594 Donkey anti-Rabbit IgG TRITC conjugated	Molecular Probes, Invitrogen	1:300	Donkey