Kaiser s glycerol gelatine

To assess the effect of UV irradiation on skin cells, 6-4PP and CPD were examined immunohistochemically on 1–2 µm paraffin sections as previously described²⁴ . The sections were incubated with either anti-6-4PP (clone 64M-2, Cosmo Bio) or anti-CPD (clone TDM-2, Cosmo Bio). Alkaline Phosphatase/RED, Rabbit/Mouse (Agilent Technologies) was employed for the detection of 6-4PP⁺ and CPD⁺. Nuclei staining was performed with hematoxylin (Merck Millipore) and slides were coverslipped with Kaiser’s glycerol gelatine (Merck Millipore). Analysis of apoptosis in irradiated cells was performed by cleaved caspase-3 staining (clone 5A1E, Cell Signaling Technologies) on selected sections^{31 (link)}. Negative controls were performed by omitting the primary antibody. An AxioImager Z1 microscope (Carl Zeiss MicroImaging, Inc.) was used for histologic documentation in a blinded manner.

Desiccated C. papaya seeds were imbibed for 5 d on 1% agar-water and then heat-shocked at 35 °C for 4 h. Imbibed (non-germinating) and cracked (germinating) seeds were selected to observe the changes occurring in the seed coat. Individual seeds were encased in Jung tissue freezing medium (Leica Instruments GmbH) and sectioned using a Leica CM3050 S cryostat microtome. Sections (20 µm thick) were adhered to glass slides prepared with Kaiser’s glycerol gelatine (Merck) and freeze-dried overnight at –20 °C. Slides were desiccated using a series of ethanol dilutions (50%, 70%, and 100%), covered with a drop of Histoclear (Sigma) to exclude air bubbles and mounted with Histomount (Sigma). Slides were studied on a Stemi SV11 (Zeiss) microscope and images were captured with a colour Axiocam (Carl Zeis Ltd.). Representative images were taken corresponding to transverse sections through the top (micropylar and embryo radicle end) and middle (embryo hypocotyl region) of the seed.

For the evaluation of adipogenic differentiation, samples from two dogs were investigated. ASCs were divided into three groups: labelled, unlabelled and negative control cells. Both labelled and unlabelled cells were incubated in adipogenic medium (DMEM low glucose, Gibco life technologies), 10% FBS (PAA), 1% penicillin/streptomycin (AppliChem), 0.1 μM dexamethasone (Sigma Aldrich), 5 μg/mL ITS (Sigma Aldrich), 0.2 mM indomethacin (Sigma Aldrich) and 0.5 mM IBMX (Sigma Aldrich) for 2 weeks, while negative control cells were incubated in the standard medium. After this time the cell population was fixed in 4% PF and red oil O staining was performed. The nuclei were stained with hematoxyline (Merck) for 10 s. The glass slides with the stained cells were embedded in Kaiser’s glycerol gelatine (Merck) and examined by light microscopy (Leica camera 090135006; Leica Microsystems).

SGHPL-4 cells were seeded in chamber slides (8 × 10⁴ cells/chamber) and cultured for 48 h. Thereafter, cells were fixed in acetone for 10 minutes and rehydrated in PBS for 5 minutes. Cells were stained using the UltraVision Large Volume Detection System HRP Polymer Kit (Thermo Fisher Scientific, Carlsbad, CA, USA), as previously described [55 (link)]. In brief, endogenous peroxidase was blocked with UltraVision hydrogen peroxide block for 10 min. Three washing steps with tris-buffered saline (TBS + Tween) were followed by Ultra Vision Protein Block including 10% human serum for 5 min. Polyclonal goat anti-CX3CL1 (2 μg/mL, AF365, R&D Systems) was diluted in Antibody Diluent (DAKO) and incubated on slides for 45 min at RT. After three TBS washing steps detection was achieved by incubation with HRP-conjugated rabbit anti-goat antibody (5 µg/mL, P0449, Dako, Agilent Technologies), and 3-amino-9-ethylcarbacole (AEC, Thermo Scientific), according to the manufacturer’s instructions. Nuclei were stained with hemalaun and slides were mounted with Kaiser’s glycerol gelatine (Merck). Images were acquired with a Leica microscope (Leica DM6000B) and a digital camera (Olympus DP72, Tokyo, Japan).

Immunohistochemistry was performed using the UltraVision LP Detection system (Thermo Scientific, Fremont, USA) according to the manufacturer’s instructions. Primary antibodies were diluted in antibody diluent (Dako, Vienna). Table 1 lists details of all antibodies and the appropriate immunoglobulin G (IgG) negative control antibodies used in their respective dilutions. Sections were counterstained with Mayer’s haemalaun and mounted with Kaiser’s glycerol gelatine (Merck, Vienna Austria).Table 1

Antibodies used in immunohistochemistry

Antigen/antibody	Company	Dilution	Host/isotype
Cytokeratin 7 (KRT7)	Acris (Herford, Germany)	1:1000	Rabbit IgG pc
Major histocompatibility complex, class I, G (HLA-G)	BD Biosciences (Vienna, Austria)	1:1000	Mouse IgG mc
Matrix metalloproteinase 1 (MMP-1)	Protein Tech (Rosemont, USA)	1:500	Rabbit IgG pc
Von Willebrand factor (VWF)	Sigma Aldrich (St. Louis, USA)	1:1000	Rabbit IgG mc
Mouse IgG1 (DAK-GO1)	Dako (Carpinteria, USA)	1:100	Mouse IgG mc
Rabbit immunoglobulin fraction (X 0903)	Dako (Carpinteria, USA)	1:300	Rabbit IgG mc

Serial sections from six different TissueTek-embedded biopsy samples per patient were cut with a cryostat (5 μm), fixed with acetone, and endogenous peroxidase activity blocked with 0.3 % hydrogen peroxide in 0.1 % sodium azide/phosphate-buffered saline. Sections were stained overnight at 4°C with Abs against CSF-1 (R&D), IL-34 (M4, provided by Five Prime Therapeutics Inc.) and CD68 (Dako). Equivalent concentrations of irrelevant mouse monoclonal Abs were used as negative controls. Sections were then washed and incubated with goat anti-mouse-horseradish peroxidase (HRP) (Dako), except in the case of anti-IL-34 Ab, which was followed by incubation with biotinylated tyramide and streptavidin-HRP. Sections were developed with amino-ethylcarbazole (AEC, Vector Laboratories), counterstained with Gill’s hematoxylin (Klinipath), and mounted in Kaiser’s glycerol gelatine (Merck). Stained sections were analyzed by computer-assisted image analysis using the Qwin analysis system (Leica) as previously described in detail [42 (link)]. Values of integrated optical densities (IOD)/mm² were obtained and corrected for the total number of nuclei/mm². Data were presented as the number of positive cells/mm² for quantitative analysis of cell-type-specific markers.