Alexa 488

ICAM-1 expression assay was performed as described previously [19] (link). Shortly, HUVECs (2×10⁴/ml), grown in either transparent 96-well plates and adhered for 6 h, were incubated for 1h with 25 µg/ml compound AA, BB, C, D or crude extract and stimulated with the pro-inflammatory cytokine TNF-alpha (20 ng/ml) for further 18 h. Subsequently, the level of ICAM-1 expression can be quantified by means of cell-based ELISA. Upon washing with PBS and fixing the cells for 2 min with methanol (−20°C), wells were subsequently blocked from unspecific binding with PBS+5% BSA. Subsequently, ICAM-1 expression was assessed using a monoclonal anti-human ICAM-1 (CD 54) (Clone 8.4A6) purified mouse Immunoglobulin (1 µg/ml, Sigma, Austria) or an isotype-matched control IgG (1 µg/ml, Sigma, Austria) as the primary antibody and a goat anti-mouse polyclonal antibody labelled with Alexa-488 (1 µg/ml, Sigma, Austria) as a secondary antibody. After washing, the fluorescence was then detected in the multiplate Reader (EX/EM 485/535). The % of ICAM-1 upregulation was then calculated as follows: % of basal ICAM-1  =  (Test Signal –Control antibody signal)/(Basal signal – control antibody signal) x 100%.

Conjugation of protein or nanobody was performed with an eightfold (laminin; Roche, 11243217001) or fourfold (R2-myc-his; a gift from S. Oliveira) molar excess of NHS ester (ATTO 425, ATTO 647N; ATTO-TEC, Alexa 488, Alexa 594; Sigma-Aldrich) in a minimal volume (<50 µl) of Milli-Q water (MQ). Protein/nanobody and NHS ester were mixed by vortexing and incubated overnight at 4 °C before storing 10-µg aliquots at −20 °C. Fibrinogen pre-conjugated with Alexa Fluor 647 (Invitrogen, F35200) was reconstituted in 0.1 M sodium bicarbonate buffer, pH 8.3, and aliquots were stored at −20 °C.

Transverse serial sections of FDP muscle (10 μm thick) were obtained using a cryostat maintained at − 25 °C (HM-560, Microm H, Thermo Scientific, Waltham, Massassuchets, USA). Sections were stored at − 20 °C until histochemical staining. Before labeling, the sections were dried and fixed for 10 min in acetone. The sections were then washed in PBS, blocked and permeabilized with PBS 0.1% Triton X-100 and 20% horse serum.
For cross-sectional area (CSA) determination, the sections were incubated for 1 h with a rabbit anti-laminin antibody (1/400) (Sigma Aldrich, Saint-Louis, Missouri, USA), washed and then incubated with a secondary antibody conjugated to ALEXA 488 (goat anti-rabbit, Sigma, 1:800).
For muscle fiber typing determination, the sections were incubated with anti-MHC primary antibodies (anti-slow (I) MyHC, BA-D5, Developmental Studies Hybridoma Bank, 1:10 and anti-fast (II) MyHC, M4276, Sigma-Aldrich, 1:200) for 1 h at 37 °C, followed by washes in PBS and incubation with the secondary antibodies (1/800) (ALEXA 488; ALEXA 568, A11031, Invitrogen, Carlsbad, California, USA,) for 1 h.
The sections were scanned using a Nanozoomer (Hamamatsu), and CSA and fiber typing and were determined using ImageJ® software (version 1.46r).

Free-floating sections were washed in TBS, blocked with 5% bovine serum albumin (BSA; New England BioLabs, Ipswich, MA, USA) containing 0.1% Triton X-100 for 1 h at room temperature to reduce non-specific staining and incubated in primary antibody solution containing 2% BSA, 0.25% Triton X-100, 0.1% NaN₃ in 0.1 M TBS for 48 h at room temperature. The following primary antibodies were used: anti-βIV-spectrin (rabbit, 1:500; selfmade, see Schlüter et al., 2017 (link)), anti-synaptopodin (rabbit, polyclonal, 1:1,000; Synaptic Systems, Göttingen, Germany) and anti-GFP488 (mouse, 1:500, fluorescence-labeled Alexa 488; Sigma-Aldrich). For immunofluorescence detection, sections were incubated with secondary fluorescence-labeled antibodies (1:1,000; Alexa 488, 568, 633; Vector Labs., Burlingame, CA, USA) for 24 h at room temperature.

Macrophages positive for phosphorylated SMAD2, indicative of TGFB signalling [31 (link)], were detected by immunofluorescent staining of the formalin-fixed paraffin-embedded mammary glands. Following antigen retrieval, F4/80 antibody conjugated to Alexa-750 (Caltag Laboratories, Burlingame, CA, USA) and rabbit anti-pSMAD2 monoclonal antibody (Invitrogen) both at a 1:100 dilution were incubated with the slides overnight at 4 °C. Goat anti-rabbit antibody conjugated to Alexa-488 was used to detect pSMAD2 (1:500 dilution; 1 h at 4 °C) followed by 4′, 6-diamidino-2-phenylindol (DAPI) (Sigma, St Louis, USA) staining for 10 min at 1:1000 dilution. Images were captured and collected using an Olympus FV3000 Confocal Microscope at × 40 magnification. Macrophages were selected randomly for imaging, and the number of macrophages positive for pSMAD2 quantified and expressed as percent positive per mammary gland.