Biotinylated secondary antibody

Immunohistochemistry using sections of paraffin-imbedded tumors was carried out as previously described [30] (link). Briefly, after antigen retrieval (microwave, citrate buffer, ph = 6), mouse anti-YKL-40 or corresponding isotype control (see above) was used as first antibody, respectively, followed by incubation with biotinylated rabbit anti-mouse (Dako, Glostrup, Denmark) and visualization using the streptavidin-alkaline-phosphatase based Vectastain ABC kit (Vector, Burlingame, CA, USA). Slides were scanned by a Mirax microscope (Zeiss, Jena Germany) and the Mirax Viewer (Zeiss) software was used to take images. Other antibodies used for immunohistochemistry (with corresponding biotinylated secondary antibodies (all Dako)) were: Anti murine EDG-1 (S1P1) (Santa Cruz, Heidelberg, Germany), anti-murine CD45 (clone 30-F11, BD) and anti-human Ki-67 (clone MIB-1, Dako). Images of four randomly chosen fields of vision (not containing necrotic areas) were taken with the viewer software. Human Ki-67 or murine CD45 positive cells were counted and murine S1P1 positive areas quantified using the ImageJ software (Wayne Rasband, NIH, USA). Positive cells or percent positive area per field of vision were calculated for each tumor.
Histochemical Masson-Goldner staining of paraffin-imbedded tumors was performed as described [33] .

The spleen and liver was removed, fixed in 10% neutral-buffered formalin solution, embedded in paraffin, and cut into tissue sections, which were stained with hematoxylin and eosin. Paraffin-embedded sections were deparaffinized and immersed in 80 °C water bath in 10 mM sodium citrate buffer with 0.1% Tween 20 overnight for antigen unmasking. Slides were incubated with primary antibody against CSF2RA (1:100, Biolegend), CSF2RB (1:80, Lifespan Bioscience), GM-CSF (1:150, Novus Biologicals), P-ERK1/2 (1:400, Cell Signaling), E-cadherin (1:100, Cell Signaling) or Fibronectin (1:100, Cell Signaling) in PBS containing 1% BSA and 10% goat serum. Biotinylated secondary antibodies (Dako) were added and incubated at room temperature for 1 hour. Streptavidin-HRP (BD Pharmingen) was added, and after 40 min the sections were stained with DAB substrate and counterstained with hematoxylin.

At the experimental endpoint at day 7, the entire wound was paraffin-embedded. At experimental endpoint at day 29, the wound area was cut into two halves. One half was paraffin embedded. The other half was used to measure the breaking strength. Paraffin-embedded sections (5 µm), n = 6 per group and time point, were deparaffinized and rehydrated. Antigen retrieval was performed in Tris-EDTA buffer containing 0.1% trypsin (Invitrogen, Carlsbad, CA). Endogenous peroxidase activity was quenched by exposing to 0.1% hydrogen peroxide in PBS containing 0.1% Tween 20 (PBST). After blocking with 4% nonfat milk powder in PBST, the sections were incubated with mouse anti-CD68 (1∶100; AbD Serotec, Düsseldorf, Germany) and goat anti-CD34 (1∶200; DakoCytomation, Glostrup, Denmark), respectively, followed by incubating with the corresponding biotinylated secondary antibodies (DakoCytomation). The antigen-antibody complex was detected by streptavidin-peroxidase (DakoCytomation) and 3,3′-diaminobenzidine (Sigma-Aldrich, Zwijndrecht, the Netherlands).

Liver tissue sections were deparaffinized and rehydrated with xylene and decreasing graded ethanol, whereas antigen retrieval was engendered by heating the sections in 10 mM sodium citrate buffer (pH = 6) in a microwave for 10 min sub boiling. Slides were blocked for nonspecific binding with 3% H₂O₂ for 10 min, avidin-biotin (DAKO, Hamburg, Germany), followed by 5% serum of the species of which the second antibody was made in 1% bovine serum albumin (BSA), and 0.05% Tween 20 in phosphate-buffered saline (PBS). Primary antibodies were diluted in 1% BSA in PBST to concentrations of 2–5 μg/mL and incubated at 4 °C overnight, while omitting primary antibody was used for negative control. Sections were incubated with biotinylated secondary antibodies (DAKO), followed by avidin-biotin conjugated peroxidase (VECTASTAIN^® Elite ABC-HRP peroxidase kit (PK-6100), Vector Laboratories, Burlingame, CA, USA) and 3,3′-diaminobenzidine substrate (SIGMAFAST™, Sigma-Aldrich, Taufkirchen, Germany, #D4293).

We employed a standardized immunohistochemistry protocol (Peixoto-Santos et al., 2015 (link)) to estimate NeuN+ neuronal density and to evaluate parvalbumin-expressing (PV) interneurons, mGluR5 expression, and glial fibrillary acid protein (GFAP) reaction. Briefly, endogenous peroxidases were blocked and the antigens were exposed by microwave-induced retrieval. We used the primary antibodies against NeuN (Chemicon, USA; 1:1000), PV (Calbiochem, USA; 1:500), mGluR5 (Millipore, USA; 1:200), and GFAP (Dako, Denmark; 1:500) proteins, and biotinylated secondary antibodies (Dako, Denmark; 1:200). Revelation was performed with Elite ABC Kit (Vector) and diaminobenzidine solution (Pierce).
Images from the regions of interest were captured at 200x magnification in the Axio Scope.A1 microscope system (Carl Zeiss). The regions of interest included the hippocampus (CA1 and GL, granule layer of dentate gyrus), entorhinal cortex (EC), infralimbic (IL) and prelimbic cortex (PL), and thalamic reticular nucleus (TRN). Images were then processed in Image J software (NIH, USA; 1.48 v). Data are shown as the density of NeuN positive cells and immunopositive area for PV, mGluR5, and GFAP.

The collagen-CAs-collagen ‘sandwiches’ were fixed in 4% paraformaldehyde for 24 hours, paraffin-embedded, longitudinally sectioned and stained with H&E. Other sections were incubated with primary antibodies follows anti-ALP (1:200, Abcam, British), anti-periostin (1:200, Santa, USA), anti-fibronectin (1:200, Abcam, British), anti-Runx2 (1:200, Abcam, British) and anti-integrin β (1:200, Abcam, British). PBS was used for the negative controls instead of the primary antibodies. Bio-tinylated secondary antibodies (1:1000) were purchased from Dako (Dako, USA). The staining sections were observed with a light microscope (Nikon, Japan).