Mayer s haematoxylin

4% PFA fixed and paraffin-embedded thymi were sliced into 8 μm tick sections and stained with antibodies. The sections were counterstained with Mayer’s haematoxylin (Merck) and mounted in Entellan (Merck). Haematoxylin and Eosin (H/E) staining was performed according to standard protocol. Images were acquired with the light microscope Axioskop (Zeiss) and analyzed with the Adobe Photoshop software.

Cytospins of SVF cells isolated from MAT and SAT of mice sacrificed
24 h after infection with N. caninum were prepared as
follows. The slides were methanol fixed and specifically stained for N.
caninum by a previously described protocol11 (link). Briefly,
peroxidase activity was blocked by treatment with 0.3% hydrogen peroxide in
methanol (Merck, Darmstadt, Germany) for 20 min. Sections were then
incubated in a moist chamber for 20 min with normal rabbit serum
(Dako, Glostrup, Denmark) diluted 1:5 in 10% BSA (Sigma), to eliminate
non-specific staining. Excess serum was removed and the sections were incubated
at room temperature, 1h45 min with goat anti-N. caninumpolyclonal serum (VMRD, Pullman, WA) diluted 1:1500. Sections incubated with
anti-N. caninum antibody were washed and incubated for
30 min at room temperature with the peroxidase-labeled rabbit
anti-goat secondary antibody (Millipore, Billerica, MA, USA) diluted 1:500. The
colour in all sections was developed by incubation with
3,3′-diaminobenzidine (Dako). After counterstaining tissue sections
with Mayer’s Haematoxylin (Merck), slides were mounted in Entellan
(Merck). A positive reaction was indicated by the presence of brown cytoplasmic
staining.

Native and decellularized lung tissue samples were fixed with 3.7%
formaldehyde solution (EMS) at 4 °C overnight. Fixed tissues
were immersed in 30% sucrose overnight and then embedded in OCT (Tissue-Tek)
and snap-frozen. 10 μm sections were obtained with a cryostat
and mounted on glass slides. For DNA staining with Hoechst, slides
were hydrated with PBS and stained for 15 min in 1 μg/mL Hoechst
solution (Invitrogen) and visualized by fluorescence microscopy. Haematoxylin
and eosin staining was performed to validate the absence of nuclei
upon decellularization. Briefly, slides were hydrated and stained
with Mayer’s haematoxylin (Merck) for 3 min, followed by a
3 min wash with tap water. Then, slides were immersed in 95% ethanol
and stained with eosin solution (bright-slide) for 45 s. For collagen
staining, Sirius Red (PolySciences) in a saturated aqueous solution
of picric acid was used. Slides were immersed in Sirius Red solution
for 1 h and then rinsed in 0.5% acetic acid solution. Alcian blue
staining was performed to visualize sGAGs. Slides were hydrated and
stained with 1% Alcian blue (Sigma) in 3% acetic acid solution pH
2.5 for 30 min, followed by a 2 min wash with tap water. After staining,
all slides were dehydrated with graded alcohol, mounted, and visualized
by light microscopy.

Serial frozen sections of wound tissue were fixed in dried acetone (Fisher, UK) before being hydrated in tris buffered saline (TBS) wash buffer and incubated in a 10% horse serum wash buffer for one hour. Anti-HGFL (R&D 352-MS) or anti-RON (R&D AF691) primary antibodies were added to the sections at a dilution of 1:100 and incubated for 1 h. The sections were then washed in TBS wash buffer 4 times and antibody localization was identified with a standard streptavidin-biotin peroxidase technique (Vector Laboratories, Peterborough, UK). The final reaction product was developed with 3,3'-diaminobenzidine (DAB), and then the sections were counterstained with Mayer’s haematoxylin (Merck, Germany), washed in running tap water, dehydrated through a graded series of alcohol, cleared in xylene, mounted in DPX mounting medium (Merck, Germany) and observed under a microscope. Any positive staining was seen as a brown-black deposit, whilst negative areas were identified as blue counterstained nucleated cells.

Gerbils at the age of 21 days were infected with strain CA16-194 at a TCID₅₀ of 10^5.5 and a 100 × LD₅₀. After four days infection, the gerbils were sacrificed and subjected to histopathologic and immunohistochemical examination. Tissue samples (skeletal muscle, spinal cord, and brainstem) of the gerbils were obtained immediately after anesthetization, fixed in 10% neutral buffered formalin for two days, dehydrated in ascending concentrations of ethanol and embedded in paraffin. For each tissue specimen, several sections were sliced, mounted on poly-L-lysine-coated slides and stained with haematoxylin and eosin or with Nissl before histopathologic examination. For immunohistochemical testing, tissue sections were dewaxed, dehydrated, and incubated with 0.3% H₂O₂ in PBS for inhibiting endogenous peroxidase activity. Polyclonal rabbit anti-CA16 antibodies (1:100 dilution), produced by immunization of rabbits using whole inactivated CA16 virus, were added and samples were incubated for 1 h at room temperature. A peroxidase-conjugated anti-rabbit antibody (1:200 dilution; Cell Signaling Technology, Beverly) was added for 30 min at room temperature. Viral antigens in the tissue sections were visualized by incubation with the peroxidase stain DAB (Dako, Glostrup, Denmark), which was followed by counterstaining with Mayer’s haematoxylin (Merck, Darmstadt, Germany).

Immunohistochemistry for ZEB1 on 97 formalin-fixed, paraffin-embedded samples of different histological types of breast cancers from patients who underwent surgery was done as previously described17 (link). In brief, sections (4 μm) were deparaffinized, rehydrated and pretreated in a pressure cooker (or in a microwave) in 10 mM citrate buffer pH 6,0. They were then incubated with a polyclonal antiserum against ZEB1, diluted 1:800 (Sigma, #HPA027524) at 4 °C overnight. Slides were washed three times with TBS/0,05% Tween20 and developed with the EnVision-System (DAKO, #K4003) according to the manufacturer's protocol. Finally they were counterstained with Mayer's haematoxylin, diluted 1:10 (Merck; #1.09249.0500) for 60–90 s. For detection of CTGF and YAP1 expression, samples were stained with rabbit anti CTGF (Abcam ab6992, diluted 1:150) and rabbit anti YAP1 (Proteintech #13584-1-AP, diluted 1:400). Sections were masked and analysed independently by two researchers. Samples were retrieved from local archives and usage was approved by the Ethics Committee of the University of Erlangen-Nuremberg (no. 374–14 Bc). Informed consent was provided by the patients when required.

In kidneys, scheduled for histopathology, dehydration and embedding in paraffin was performed. Kidney tissue was cut and sections were transferred on glass slides. To assess glomerulosclerosis periodic acid–Schiff (PAS) staining was performed. Briefly, sections were deparaffinized in Xylene and subsequently rehydrated in a descending ethanol series. After incubation for 10 min each in 0.9% periodic acid (Carl Roth, Karslruhe, Germany), Schiff reagent (Merck, Darmstadt, Germany) and Mayer’s haematoxylin (Merck) the sections were dehydrated in an ascending ethanol series and xylene, and covered with HistoMount (National Diagnostics, Atlanta, USA). A NanoZoomer S360 (Hamamatsu, Geldern, Germany) was used to acquire images. To determine the extend of glomerular damage, the number of crescentic glomeruli was counted in a blinded manner. Crescents were defined as two or more cellular layers in the Bowman`s space. A minimum of 50 glomeruli were assessed per kidney^{18 (link)}.