Immunostaining was applied on paraffin and cryostat sections. Paraffin sections were treated by microwave for 20 min at 400W in citrate buffer (pH 6.0) after deparaffinization and hydratation. Five μm thick frozen sections cut from each tissue were fixed in acetone for 10 min, air-dried at room temperature for 1 hour. After antigen retrieval both frozen and paraffin samples were incubated for 1 hour at room temperature with primary antibody: NCAM clone Eric-1 (1:100, Ancell Corporation, USA), NCAM clone 123C3.D5 (LabVision, USA), FGFR1 clone M19B2 (1:100, Abcam, UK), HE4 (1:40, ab24480, Abcam, UK), integrin α5β1 clone SAM-1 (1:200, Chemicon Europe). NCAM/Eric-1 was used on cryostat samples, while paraffin samples were incubated with NCAM/123C3.D5. The EnVision staining method (DAKO, Denmark), visualization of antigen-antibody reaction by 3,3'-diaminobenzidine (DAB) and subsequent counterstaining with hemalaun (Merck, USA), were conducted. Controls were performed as previously described [2 (link)], and for mouse monoclonal antibodies as isotype control mouse IgG1 (ab91353, Abcam, UK) antibody was also used. Slides were evaluated using the light microscope BX53 with DP12 CCD camera (Olympus, Germany).
Hemalaun
Manufactured by Merck Group
Sourced in Germany
Hemalaun is a laboratory staining solution used for the histological staining of biological samples. It is a hematoxylin-based stain that primarily stains cell nuclei, providing contrast to aid in the visualization and analysis of cellular structures.
Automatically generated - may contain errors
Lab products found in correlation
Alcian blue, by Merck Group (4 mentions)
Dab kit, by Merck Group (4 mentions)
Advance hrp link, by Agilent Technologies (3 mentions)
Advance hrp enzyme, by Agilent Technologies (3 mentions)
Aquatex, by Merck Group (3 mentions)
Deadend colorimetric tunel system, by Promega (3 mentions)
Chondroitinase abc, by Merck Group (2 mentions)
Non immune igg, by Merck Group (2 mentions)
Roti histofix formaldehyde fixation reagent, by Carl Roth (2 mentions)
Monoclonal anti col 2 antibodies, by OriGene (2 mentions)
Dp12 ccd camera, by Olympus (1 mentions)
Ab24480, by Abcam (1 mentions)
Ab91353, by Abcam (1 mentions)
Image pro 10, by Media Cybernetics (1 mentions)
Trypsin digestion, by Merck Group (1 mentions)
Cellsens standard 1, by Olympus (1 mentions)
X tra adhesive precleaned micro slides, by Leica (1 mentions)
Mouse monoclonal anti aβ antibody clone 6e10, by BioLegend (1 mentions)
Colorview soft imaging system, by Olympus (1 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (1 mentions)
20 protocols using hemalaun
1
Immunostaining Protocol for NCAM, FGFR1, HE4, and Integrin α5β1
2
Histology and Immunohistochemistry of Aggregates
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For histology, aggregates were fixed in 4% paraformaldehyde for 1 h, followed by dehydration in graded alcohols, paraffin embedding, sectioning to 4 μm, and staining with haematoxylin/eosin (H&E) or alcian blue (Sigma) as described previously [7 (link), 8 (link)]. For visualisation of ALP activity, a histochemical assay was performed according to the instructions of the supplier (Sigma).
Immunohistochemistry on alternate sections was performed as described previously [7 (link)]. Briefly, following the respective pre-treatments with pepsin (1 mg/mL), or chondroitinase ABC (Sigma; 5 U/mL), or trypsin (0.25%) sections were incubated overnight with the following primary antibodies: monoclonal anti-COL type II (Acris Antibodies GmbH, Hiddenhausen, Germany), anti-chondroitin-4-sulphate (CS4) (Millipore GmbH, Schwalbach, Germany) or anti-collagen type X (COL type X) (Calbiochem, Bad Soden, Germany). Immunohistodetection was performed by treatment with Advance™ HRP link and Advance™ HRP enzyme (Dako, Hamburg, Germany) followed by diaminobenzidine staining (DAB kit; Sigma), and slides were finally counterstained with hemalaun (Merck, Darmstadt, Germany). In addition, controls with non-immune Ig G (Sigma) instead of the primary antibodies were also performed.
Immunohistochemistry on alternate sections was performed as described previously [7 (link)]. Briefly, following the respective pre-treatments with pepsin (1 mg/mL), or chondroitinase ABC (Sigma; 5 U/mL), or trypsin (0.25%) sections were incubated overnight with the following primary antibodies: monoclonal anti-COL type II (Acris Antibodies GmbH, Hiddenhausen, Germany), anti-chondroitin-4-sulphate (CS4) (Millipore GmbH, Schwalbach, Germany) or anti-collagen type X (COL type X) (Calbiochem, Bad Soden, Germany). Immunohistodetection was performed by treatment with Advance™ HRP link and Advance™ HRP enzyme (Dako, Hamburg, Germany) followed by diaminobenzidine staining (DAB kit; Sigma), and slides were finally counterstained with hemalaun (Merck, Darmstadt, Germany). In addition, controls with non-immune Ig G (Sigma) instead of the primary antibodies were also performed.
3
Immunohistochemical Analysis of Protein Expression
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T-47D, MCF-7 and BT-20 cells were fixed subsequent to experimental treatment using ROTI® Histofix formaldehyde fixation reagent (4% formaldehyde in PBS, pH 7, Carl Roth, Karlsruhe, Germany). An indirect immunoperoxidase technique was applied to visualize the target protein expression. Antigen retrieval was performed in a 10 mmol/L sodium citrate buffer (pH 6.0) followed by inhibition of endogenous peroxidase by incubation for 30 min with 3% H2O2. After two washes in trissaline buffer, slides were incubated with 1% goat serum for 30 min to block unspecific staining. Following overnight incubation with the target protein antibody (Supplementary Table 3) at RT and two washing steps with PBS, slides were exposed to ImmPRESS peroxidase polymer reagent (BIOZOL, Eching, Germany) for 60 min at RT. Staining was achieved by 3,3-diaminobenzidine (DAB; BIOZOL, Eching, Germany) and the cells were counterstained with hemalaun (Merck). The colour intensity was quantified and evaluated with the software Image-Pro® 10 (MEDIA Cybernetics, Rockville, USA), according to the manufacturer’s specifications. For better comparison, the percentage staining in comparison to the cell surface was calculated. A staining below 5 % of the area was considered negative.
4
Apoptosis Detection in Tumor Samples
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To detect DNA fragmentation and consequently apoptotic cells in tumor specimens, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay (TUNEL; DeadEnd™ Colorimetric Tunel System, Promega) was used according to the manufacturer’s instructions. In brief, formalin-fixed and paraffin-embedded slides (3 μm) were deparaffinized by Xylol, hydrogenated through a graded ethanol series, 0.85% NaCl, and PBS buffer, and fixed with 4% paraformaldehyde. After covering the tissues with protein kinase K, they were incubated with the reaction mixture containing terminal deoxynucleotidyl transferase, equilibration buffer, and biotinylated nucleotide mix for 1 h at 37 °C to label 3ʹOH DNA ends. Streptavidin- and DAB-bound biotin was quantified, and nuclei were stained dark brown. Slides were counterstained with hemalaun (Merck Millipore, Darmstadt, Germany) and covered with aquatex (Merck Millipore, Darmstadt, Germany). DNA fragmentation was detected by selecting four fields at ×40 magnification in each tumor section at the invasive front and counting positive stained nuclei. Data were expressed as the number of apoptotic cells per high-power field.
5
Histological and Immunohistochemical Analysis of Bone Tissue
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For light microscopy, formalin-fixed specimens were embedded in paraffin. 5 µm thick sections were cut and stained with hematoxylin and eosin (H&E) according to standard procedures and examined by light microscopy (DM4000B Leica Mikrosysteme, Wetzlar, Germany).
For the immunohistochemical analysis, formalin-fixed specimens were embedded in paraffin and cut into 5 µm thick sections. The following antibodies were used as primary antibodies: rabbit anti-collagen type I (1:800, BIOLOGO, Kronshagen, Germany), rabbit anti-collagen type IV (1:400, Acris Antibodies GmbH, Hiddenhausen, Germany), mouse anti-osteocalcin (1:200, QED Bioscience Inc., San Diego, USA) and mouse anti-SPARC (1:200, Santa Cruz Biotechnology, Santa Cruz, USA). A biotin-conjugated goat anti-rabbit antibody (1:600, Dianova, Hamburg, Germany) or a biotin-conjugated goat anti-mouse antibody (1:200, Dianova, Hamburg, Germany) served as secondary antibodies. Incubation with streptavidin-horseradish peroxidase (Dianova, Hamburg, Germany) was followed by color development with aminoethylcarbazole (AEC) substrate (Axxora Deutschland GmbH, Loerrach, Germany) at room temperature. Color development was stopped under microscopic examination by washing with water. The sections were counterstained with hemalaun (Merck, Darmstadt, Germany) and examined by light microscopy (DM4000B Leica Mikrosysteme, Wetzlar, Germany).
For the immunohistochemical analysis, formalin-fixed specimens were embedded in paraffin and cut into 5 µm thick sections. The following antibodies were used as primary antibodies: rabbit anti-collagen type I (1:800, BIOLOGO, Kronshagen, Germany), rabbit anti-collagen type IV (1:400, Acris Antibodies GmbH, Hiddenhausen, Germany), mouse anti-osteocalcin (1:200, QED Bioscience Inc., San Diego, USA) and mouse anti-SPARC (1:200, Santa Cruz Biotechnology, Santa Cruz, USA). A biotin-conjugated goat anti-rabbit antibody (1:600, Dianova, Hamburg, Germany) or a biotin-conjugated goat anti-mouse antibody (1:200, Dianova, Hamburg, Germany) served as secondary antibodies. Incubation with streptavidin-horseradish peroxidase (Dianova, Hamburg, Germany) was followed by color development with aminoethylcarbazole (AEC) substrate (Axxora Deutschland GmbH, Loerrach, Germany) at room temperature. Color development was stopped under microscopic examination by washing with water. The sections were counterstained with hemalaun (Merck, Darmstadt, Germany) and examined by light microscopy (DM4000B Leica Mikrosysteme, Wetzlar, Germany).
6
Immunohistochemical Analysis of EpCAM
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To verify the EpCAM expression, two TMAs consisting of 55 primary lung tumour tissues and 76 lung cancer brain metastases were stained for EpCAM by IHC. In addition, the tissue from nine matching primary tumour and brain metastasis samples was analysed. The tissue slides were de-paraffinized with xylol and rehydrated with an ethanol series followed by a 10 min trypsin digestion step (37 °C, Sigma-Aldrich). Unspecific binding was blocked using DAKO ChemMate peroxidase blocking solution (Dako, Denmark) and the tissue was incubated with the primary EpCAM antibody (1:75, NCL Esa antibody; Novocastra, UK) for 60 min at room temperature. For visualization, the DAKO Envision kit was used with DAKO DAB solution (Dako Denmark A/S, Glostrup, Denmark) and hemalaun (Merck, Germany) as counterstain. The tissue was analysed blinded for signal intensity by two independent researchers (AH, HW). Both signal intensity and signal distribution were multiplied to a final value and grouped according to a threshold of ≤0.5 into negative, ≤2 into intermediate and ≥2 into strong stainings.
7
Quantifying Amyloid-Beta and Microglia in Mouse Hippocampus
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Brain tissue was fixed in 4% phosphate-buffered formalin, embedded in paraffin and sliced in 4 µm-thin sections. The sections were put on X-tra Adhesive Precleaned Micro Slides (Leica, Wetzlar, Germany) and exposed to mouse monoclonal anti-Aβ antibody (clone 6E10; 1:1000, BioLegend, San Diego, CA, USA, as described by the authors of [28 (link)]) and goat polyclonal anti-iba1 antibody (1:1000, Abcam, Berlin, Germany). DAB chromogen Universal LSAB® kits (System-HRP; DakoCytomation, Dako, Jena, Germany) were used for development according to the manufacturer’s instructions. The sections were counterstained with hemalaun (Merck, Darmstadt, Germany), and images were acquired on microscope type BX51 with a Color View Soft Imaging System and the corresponding software cellSens Standard 1.14 (all from Olympus, Hamburg, Germany). Appropriate negative staining images are provided in Appendix A (Figure A1 ). Within the hippocampus (n = 5–10 of each mouse strain and feeding), the number and the area of anti-Aβ positive plaques, as well as the number of iba1-positive cells, were assessed and measured semiautomatically with ImageJ 1.47 v. in a high-power field (HPF) and are given in n/HPF and µm2 for the area.
8
SNCG Protein Expression Immunodetection
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Cell lines MFE-296, EFE-184, ISHIKAWA and An3-Ca were fixed after experimental treatment using ROTI® Histofix formaldehyde fixation reagent (4% formaldehyde in PBS, pH 7, Carl Roth, Karlsruhe, Germany). Anti-gamma-Synuclein B-21 rabbit antibody (sc-135676, SCBT) was used for detection of SNCG protein. For visualization of SNCG protein expression indirect immunoperoxidase technique was applied. Antigen retrieval was performed in a 10 mmol/L sodium citrate buffer (pH 6.0) followed by inhibition of endogenous peroxidase by incubation for 30 min with 3% H2O2. Endogenous avidin–biotin was blocked by the use of a commercial biotin blocking system (Agilent Technologies, DAKO, Santa Clara, USA) for 10 min. Unspecific staining was avoided by incubation for 30 min in skimmed milk blocking buffer. Following overnight incubation with SNCG antibody (1:2000) at RT and two washing steps with PBS, slides were exposed to biotinylated anti-rabbit immunoglobulins for 60 min at RT and treated with streptavidin-peroxidase (Agilent Technologies, DAKO, Santa Clara, USA). Staining of SNCG protein was achieved by 3, 3-diaminobenzidine (DAB; Vector Laboratories Inc., Burlingame, USA) and the cells were counterstained with hemalaun (Merck, Darmstadt, Germany).
9
Quantifying Liver Lipids and Adipocyte Size
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Liver cryosections (10 µm) were stained with Sudan III (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) to identify tissue lipids. Nuclei were counterstained with hemalaun (Merck, Darmstadt, Germany). Images were taken using the Zeiss Axio Imager.A1 microscope and the amount of lipids quantified by using the Image J 1.48 software (National Institutes of Health, Bethesda, MD, USA). From each animal (n ≥ 6 in each group), 3–6 liver slices were prepared, and 10–15 microscopic fields analyzed.
To assess tissue architecture, paraffin sections (1.5 µm) of the liver and adipose tissue were deparaffinized and stained with hemalaun and eosin (HE, both Merck, Darmstadt, Germany). HE histology of visceral gonadal white adipose tissue (termed VAT in the following) was used to determine the adipocyte size as described previously [26 (link)]. Images were taken using an Olympus BX40 epifluorescence microscope.
To assess tissue architecture, paraffin sections (1.5 µm) of the liver and adipose tissue were deparaffinized and stained with hemalaun and eosin (HE, both Merck, Darmstadt, Germany). HE histology of visceral gonadal white adipose tissue (termed VAT in the following) was used to determine the adipocyte size as described previously [26 (link)]. Images were taken using an Olympus BX40 epifluorescence microscope.
10
Histological and Immunohistochemical Analysis
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For histological analyses, aggregates were fixed in 4% paraformaldehyde for one hour afterward dehydrated in graded alcohols, embedded in paraffin and sectioned to 5 μm. Representative sections were stained using haematoxylin and eosin (H&E) for evaluation of cellularity and alcian blue (Sigma) for the detection of matrix proteoglycan.
For immunohistochemical analyses sections were washed for 20 min in Tris-buffered saline (TBS) and incubated in 5% bovine serum albumin (BSA) (Sigma). Following washing in TBS, sections were pre-digested with pepsin at 1 mg/ml in Tris–HCl (pH 2.0) for 15 min at room temperature. Sections were then incubated overnight at 4°C with a monoclonal anti-COL II primary antibody (diluted in 0.5% BSA, Acris Antibodies GmbH) for detection of collagen type II (Collagen II). Immunostaining was visualized by treatment with peroxidase-conjugated antibodies (Dako) followed by diaminobenzidine staining (DAB kit; Sigma). The slides were finally counterstained with hemalaun (Merck).
For immunohistochemical analyses sections were washed for 20 min in Tris-buffered saline (TBS) and incubated in 5% bovine serum albumin (BSA) (Sigma). Following washing in TBS, sections were pre-digested with pepsin at 1 mg/ml in Tris–HCl (pH 2.0) for 15 min at room temperature. Sections were then incubated overnight at 4°C with a monoclonal anti-COL II primary antibody (diluted in 0.5% BSA, Acris Antibodies GmbH) for detection of collagen type II (Collagen II). Immunostaining was visualized by treatment with peroxidase-conjugated antibodies (Dako) followed by diaminobenzidine staining (DAB kit; Sigma). The slides were finally counterstained with hemalaun (Merck).
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