Heart tissue was fixed overnight at room temperature in 4% paraformaldehyde solution (PFA; BIOSESANG, Korea). After washing the heart tissue, it was embedded in paraffin blocks. The tissues were cut in 4 μm slices using a microtome and stained with haematoxylin (Vector Laboratories, USA) and eosin (Sigma-Aldrich, USA). The sliced tissues were examined by light microscopy. The heart injury score was determined in sections containing the right and left ventricles using a semi-quantitative scale from 0 to 4, as follows: 0 = no injury, 1 = isolated myocyte injury, 2 = one focal area of injury, 3 = two or more areas of injury, and 4 = diffuse areas of damage compromising more than 50% of the myocardium.
Haematoxylin
Manufactured by Vector Laboratories
Sourced in United States, United Kingdom
Haematoxylin is a naturally occurring dye extracted from the heartwood of the Logwood tree. It is a commonly used stain in histology and cytology laboratories for the visualization of cellular structures.
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Lab products found in correlation
Hydrogen peroxide, by Thermo Fisher Scientific (2 mentions)
Immpress polymer reagent, by Vector Laboratories (2 mentions)
Vector dab, by Vector Laboratories (2 mentions)
Cytoseal 60 media, by Thermo Fisher Scientific (2 mentions)
Target retrieval solution, by Agilent Technologies (2 mentions)
Abc elite kit, by Vector Laboratories (2 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (2 mentions)
Sodium nitrite, by Fujifilm (1 mentions)
Pararosaniline hydrochloride, by Merck Group (1 mentions)
Naphthol as bi phosphate, by Merck Group (1 mentions)
L tartaric acid, by Fujifilm (1 mentions)
Haematoxylin, by Merck Group (1 mentions)
Abc solution, by Vector Laboratories (1 mentions)
Ba 2000, by Vector Laboratories (1 mentions)
Dpx mounting agent, by Merck Group (1 mentions)
Superfrost plus slides, by Avantor (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Axiocam, by Zeiss (1 mentions)
Imager m1, by Zeiss (1 mentions)
Dab substrate kit, by Vector Laboratories (1 mentions)
22 protocols using haematoxylin
1
Histological Evaluation of Cardiac Injury
2
Histological Analysis of Bone Regeneration
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To perform H&E and tartrate-resistant acid phosphatase (TRAP) staining, tissues fixed in 4% paraformaldehyde underwent decalcification in 10% ethylenediaminetetraacetic acid at 4 °C for 3 weeks. They were then embedded in paraffin after dehydration, and 4-µm-thick sections were created. Sectioning of the paraffin-embedded samples was performed along the sagittal plane.
H&E staining was performed using haematoxylin (Vector Laboratories, Inc., Burlingame, CA) for 3 minutes, followed by 10 seconds of eosin (Wako). TRAP staining was performed with Naphthol AS-BI phosphate (Sigma), sodium nitrite (Wako), 0.08 M L(+)-tartaric acid (Wako), and pararosaniline hydrochloride (Sigma) in 0.1 M of sodium acetate buffer (pH 5.0) at 37 °C in an incubator for 20 minutes, followed by nuclear counterstaining with haematoxylin. Under an Olympus light microscope (Olympus, Tokyo, Japan), TRAP-positive multinuclear giant cells adjacent to the collagen fibres were identified as osteoclasts. We counted the osteoclasts that were present in regions of bone regeneration and measured the area that consisted of new collagen (Olympus cellSens standard version 1.13; Olympus). The number of osteoclasts in the decalcified bone area (mm−2) was represented by the number of osteoclasts in the new collagen area.
H&E staining was performed using haematoxylin (Vector Laboratories, Inc., Burlingame, CA) for 3 minutes, followed by 10 seconds of eosin (Wako). TRAP staining was performed with Naphthol AS-BI phosphate (Sigma), sodium nitrite (Wako), 0.08 M L(+)-tartaric acid (Wako), and pararosaniline hydrochloride (Sigma) in 0.1 M of sodium acetate buffer (pH 5.0) at 37 °C in an incubator for 20 minutes, followed by nuclear counterstaining with haematoxylin. Under an Olympus light microscope (Olympus, Tokyo, Japan), TRAP-positive multinuclear giant cells adjacent to the collagen fibres were identified as osteoclasts. We counted the osteoclasts that were present in regions of bone regeneration and measured the area that consisted of new collagen (Olympus cellSens standard version 1.13; Olympus). The number of osteoclasts in the decalcified bone area (mm−2) was represented by the number of osteoclasts in the new collagen area.
3
Quantifying Blood-Brain Barrier Permeability
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To assess BBB permeability, brain sections were stained via indirect immunoperoxidase technique for endogenous Immunoglobulin G (IgG 1:500 Horse pAb biotin conjugated clone BA-2000 Vector). Free-floating sections were washed in PBS and mounted on to Superfrost Plus slides (VWR) in distilled water, then allowed to dry vertically overnight at 37°C. Sections were rehydrated in several changes of PBS and subjected to heat-mediated antigen retrieval in preheated Sodium Citrate pH6 buffer at 95°C for 30 minutes, and then allowed to cool at room temperature for 20 minutes. Slides were rinsed twice in PBS and then endogenous peroxidase activity was blocked by incubation in 3% H2O2 in distilled water at room temperature for 30 minutes. Sections were washed in several changes of PBS and then incubated for 3 hours at room temperature with primary antibody appropriately diluted in PBS, 0.3% Triton X and 0.1% BSA. Slides were washed thoroughly in PBS and 0.1% tween and then incubated for 1.5 hours at room temperature in ABC solution, as per manufacturer’s instructions (Vector). Colour was developed via a 5 minute incubation in diaminobenzidine tetrahydrochloride (DAB, Merck Millipore). Sections were counterstained with haematoxylin (Vector), dehydrated through alcohol, cleared in two changes of xylene and coverslipped using DPX mounting agent (Sigma-Aldrich).
4
Immunohistochemical Analysis of VEGF-A in Temporal Artery Biopsies
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Paraffin-embedded temporal artery samples were obtained from the TABUL study for five patients; three had a positive biopsy and two were control patients. Sections were cut at 4 μm and mounted on adhesive glass slides for staining. Slides were deparaffinised in xylene and rehydrated through a graded series of 100–50% ethanol. Endogenous peroxidase activity was blocked by 3% hydrogen peroxide. Heat-induced antigen retrieval for VEGF-A was performed by citrate buffer (10 mM anhydrous citric acid, 0.05% Tween 20, pH 6.0). Non-specific reactivity was blocked in buffer solution containing 3% filtered bovine serum albumin (BSA). Representative sections from each patient were incubated with 1:100 dilution of rabbit anti-human VEGF polyclonal IgG antibody (ABCAM). Negative control sections were incubated with non-immune rabbit IgG (R&D Systems). Secondary biotinylated goat anti-rabbit antibody (Vector Laboratories) was added at 1:250 dilution before incubation with avidin-biotin-peroxidase (ABC, Vectastain Elite kit, Vector Laboratories). Staining was developed with DAB substrate kit (Vector Laboratories) and counterstained with haematoxylin (Vector Laboratories). Images were captured using a microscope (Zeiss Imager M1) connected to a camera (Zeiss Axiocam). For general morphological analysis, serial sections were stained with Mayer's H&E.
5
Immunohistochemistry of TNFα, TNFAIP3, and IκBα
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Formalin-fixed tissue was bundled using methods previously described [27 (link)]. Briefly, bundles were processed, embedded in paraffin wax in the transverse orientation, and 4 µm thick tissue sections were cut by microtomy and processed for immunohistochemistry (IHC). Tissue sections were de-paraffinised and rehydrated, then treated with 1% v/v hydrogen peroxide in methanol to block endogenous peroxidases, followed by heat-induced antigen retrieval in 0.01 M citrate acid buffer (pH 6.0). Sections were then blocked with 5% v/v serum supplied by respective ImmPRESS kits (Vector Laboratories, Heyford, UK), followed by primary antibodies against TNFα (rat monoclonal antibody MP6-XT22, Abcam; Cambridge, UK), TNFAIP3 (mouse monoclonal antibody 66695-1-Ig, Proteintech; Manchester, UK), and IκBα (rabbit polyclonal antibody 9242, Cell Signaling Technology; Leiden, The Netherlands). Secondary antibodies were from ImmPRESS polymer detector kits, and for TNFAIP3, the use of a mouse-on-mouse ImmPRESS kit. Slides were counterstained with haematoxylin (Vector Laboratories).
6
Immunohistochemistry of FFPE Mouse Brain
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FFPE slides with mouse brain sections were deparaffinized and probed with primary antibodies as described before [11 (link)]. For antigen retrieval, slides receiving AT8 (1:1000; Invitrogen MN1020), AT100 (1:1000; Invitrogen MN1060), AT270 (1:1000; Invitrogen MN1050), AT180 (1:1000; Invitrogen MN1040) and glial fibrillary acidic protein (GFAP) (1:3000; Cell Signaling) antibodies were steamed in water at high pressure for 15 min, while Iba-1 (1:2000; Wako) antibody slides were steamed in citrate buffer pH 6.0 (Target Retrieval Solution, Dako). Slides were incubated in 3% hydrogen peroxide (Fisher Scientific) for 20 min to block endogenous peroxidase activity and then washed three times in PBS for 5 min each. Slides were then blocked in 2% fetal bovine serum (FBS) (Hyclone, GE) for 45 min before incubating in primary antibody diluted in block solution overnight at 4 °C. The following day, slides were washed and appropriate secondary antibody (ImmPRESS Polymer Reagent, Vector Labs) was applied for 30 min at room temperature. Following PBS washes, color was developed using 3,3’-diaminobenzidine (Vector DAB, Vector Labs) and slides were counterstained with haematoxylin (Vector Labs). Next, brain sections were dehydrated in a series of ethanol, cleared in xylene, mounted in Cytoseal-60 media (Fisher Scientific) and coverslipped.
7
Immunohistochemical Staining and Reticulin Analysis
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Immunohistochemical staining of FFPE sections were performed on consecutive 5 μm-thick sections. Tissue sections were subjected to deparaffinisation by xylene, rehydration in serial dilutions of ethanol, followed by heat-induced antigen retrieval in 10 mM sodium citrate (pH 6.0). Endogenous peroxidase was quenched by treatment with 3% hydrogen peroxide (Merck Millipore, Burlington, MA, USA) for 30 min and non-specific protein binding was blocked with 10% normal goat serum (Dako, Glostrup, Denmark) for 1 h. Sections were incubated with primary antibodies at appropriate dilutions at 4 °C overnight in a moist chamber. After incubation, sections were washed with Tris-buffered saline (TBS) for three times, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Dako) for 30 min. DAKO EnVision System (Dako) was used to detect signals from DAB chromogen substrate. Finally, sections were counterstained with haematoxylin (Vector Laboratories, Burlingame, CA) and mounted in DPX mounting solution (BDH Laboratory, UK). Primary antibodies of GFAP (#80788) and Vimentin (ab58462) were purchase from Cell Signaling Technology (Danvers, MA, USA) and Abcam (Cambridge, UK), respectively. Reticulin fibres in tissue sections were detected by reticulin silver staining (based on Gordon and Sweet’s method) according to manufacturer’s protocol (Merck Millipore).
8
Imaging Nanoparticle-Tumor Interactions
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Tumours collected for peptide and IO-NP binding and post-MRI were analysed. Tissue distribution of fluorescein (FAM)-labelled CSG, FAM-CREKA, FAM-IO-NP, FAM-CSG-IO-NP or FAM-CREKA-IO-NP were detected on 8 μm tissue cross-sections based on their fluorescence intensity and reactivity to anti-fluorescein-HRP antibody (polyclonal, GeneTex) and counter-stained with haematoxylin or methyl green (Vector Laboratories, Burlingame, CA, USA). For co-staining analysis, the following antibodies were used: anti-CD31 (390; ebioscience), anti-laminin (polyclonal; Millipore) and anti-collagen I (polyclonal; Abcam). For secondary detection, fluorescence-labelled, 594-conjugated anti-rat, rabbit or goat IgG (Life Technologies, Carlsbad, CA, USA) were used. Images were captured on a Nikon Ti-E microscope (Nikon Instrument Inc., Melville, NY, USA) or ScanScope XT (Aperio Technology, Inc., Vista, CA, USA). Image analysis and quantification were performed either using NIS software modules (version 4.0) or ImageScope version 12.1.0.5029 (Aperio Technology, Inc., Vista, CA, USA).
9
Immunohistochemical Staining Protocol
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Standard IHC staining was performed as previously described (20 (link)). In brief, after de-paraffinization and rehydration, sections were subjected to heat-induced antigen unmasking. Slides were then incubated with various primary antibodies at 4°C overnight, after blocking with 5% goat serum. Slides underwent color development with DAB (BD Pharmingen, New York, NJ, USA) and haematoxylin (Vector Laboratories, Burlingame, CA, USA) counterstaining. Images were captured with a microscope (BX51, Olympus, Tokyo, Japan) and processed with identical settings. Ten visual fields from different areas of each tumor were evaluated.
10
Immunohistochemical Analysis of Brain Samples
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Immunohistochemical studies were performed as described previously25 (link). Coronal brain sections were incubated in xylene and passed through series of graded alcohols and then subjected to antigen retrieval using the antigen retrieval solution (Vector Lab, Inc. CA). Tissue sections were incubated in 3% H2O2 solution for 20 min and then subjected to blocking using the vector lab blocking kit. Tissue sections were incubated overnight with Ki-67 (1:100), Cleaved Caspase-3 (1:200) and Bcl-2 (1:50) and then with secondary antibodies for 45 min at room temperature. Immunoreactivity was visualized by using the DAB substrate and counterstained with haematoxylin (Vector Lab, Inc. CA). The proliferative index was calculated as percentage of Ki-67-positive cells in five randomly selected microscopic fields at 20X per slide. TUNEL analysis was performed using the In situ Cell Death Detection Kit (Roche, Indianapolis, IN) as per the manufacturer’s protocol, and five randomly selected microscopic fields in each group were used to calculate the relative ratio of TUNEL-positive cells. DAPI was used to visualize the nuclei.
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