Hematoxylin and eosin (H&E) staining was done to examine histopathological changes according to general protocol. Shortly, sections of 6 µm were prepared and stained with H&E, dehydrated by a serial of ethanol and mounted with Canada balsam (Kanto Chemical, Tokyo, Japan). Histopathological analysis for lesion of the lung was semi-quantitatively assessed by a published procedure [16 (link)]. In brief, lung injury was scored according to (1) thickness of the alveolar wall; (2) infiltration or aggregation of neutrophils in airspace, the alveolar wall, or the vessel wall; and (3) alveolar congestion, and each item was graded on a four-point scale. Each component ranged from 0 to 3, with higher scores indicating more severe damage.
Canada balsam
Manufactured by Kanto Chemical
Sourced in Japan, Canada
Canada balsam is a natural resin derived from the balsam fir tree. It is a transparent, viscous liquid that is commonly used as a mounting medium in microscopy and histology. Canada balsam has a high refractive index and good optical properties, making it suitable for use in various laboratory applications.
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Lab products found in correlation
3 3 diaminobenzidine tetrachloride, by Merck Group (7 mentions)
Streptavidin peroxidase complex, by Vector Laboratories (7 mentions)
Cv acetate, by Merck Group (5 mentions)
Cresyl violet acetate, by Merck Group (5 mentions)
Epifluorescent microscope, by Zeiss (4 mentions)
Biotinylated goat anti rabbit igg, by Vector Laboratories (4 mentions)
Rabbit anti ki67 antibody, by Abcam (4 mentions)
Rabbit anti dcx, by Abcam (3 mentions)
Rabbit anti ki67, by Abcam (3 mentions)
Wiseven woc high clean air oven, by Daihan Scientific (3 mentions)
Vectastain abc, by Vector Laboratories (2 mentions)
Cv acetate solution, by Merck Group (2 mentions)
Cresyl violet acetate solution, by Merck Group (2 mentions)
Gill s hematoxylin solution, by Merck Group (2 mentions)
Eosin y solution, by Merck Group (2 mentions)
Bx53 light microscope, by Olympus (2 mentions)
Dp72 digital camera, by Olympus (2 mentions)
Normal goat serum (ngs), by Vector Laboratories (2 mentions)
Microtome, by Leica (2 mentions)
Trichrome stain kit, by Abcam (2 mentions)
51 protocols using canada balsam
1
Hematoxylin and Eosin Staining for Lung Histopathology
2
Histopathological Analysis of Kidney Lesions
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H&E staining was performed to observe the histopathological changes in the kidney according to the published protocol (33 (link)). The kidney sections were mounted on glass slides, dehydrated with ethanol after staining with H&E and mounted with Canada balsam (Kanto Chemical). Periodic acid Schiff (PAS) staining was performed according to the kit (cat. no. PAS-1-IFU; ScyTek Laboratories, Inc.) manufacturer's protocol. A Leica DM 2500 microscope (Leica Microsystems) was used to image the sections at fixed magnifications of x400 (H&E) and x1,000 (PAS), and in each group, 10 specific areas were captured. Analysis of glomerular lesions was performed according to a previously published procedure (25 (link)). The scoring was as follows: Normal, 0; <25% damage, 1; 26-50% damage, 2; 51-75% damage, 3; and 76-100% damage, 4(25 (link)). Histopathological analysis of lesions was performed according to a published procedure (34 (link)). In short, histopathological changes were evaluated by quantitative measurement of interstitial tubular injury and through counting the number of apoptotic and necrotic cells, tubular brush border loss, dilatation of tubules, cast formation and infiltration of neutrophils. The injury scoring was as follows: 0, None; 1, 0-10; 2, 11-25; 3, 26-45; 4, 46-75; and 5, 76-100% (34 (link)).
3
Histological Analysis of Kidney Injury
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The kidney tissues were perfused with 0.1 M phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde. They were further processed to embed them with paraffin and dissected into 7 µm thick slices. For the H&E staining, the sections were dehydrated with ethanol, stained with H&E, and mounted with Canada balsam (Kanto Chemical, Tokyo, Japan). For PAS staining, the PAS Stain Kit (Azer Scientific Inc., Morgantown, PA, USA) was used according to the manufacturer’s instructions. Histological glomerular injury was scored as follows according to the damaged regions by referring to previously established procedures [27 (link)]: 0: normal; 1: less than 25%; 2: between 25% and 50%; 3: between 50% and 75%; and 4: between 75% and 100%.
4
Immunohistochemical Analysis of Cerebral Ischemia
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To examine neuronal damage, the number of activated astrocytes and microglia in the CA1 region after induction of cerebral ischemia, immunohistochemistry was performed following an established technique [20 (link)]. In brief, the sections were immersed in 0.3% H2O2 for quenching. Then, the tissue sections were blocked with 5% goat serum and horse serum. Next, tissue sections were incubated with diluted mouse anti‐NeuN (1:800, Merck Millipore, MA, USA #cat MAB377), rabbit anti-GFAP (1:1,000, GeneTex, Irvine, CA, USA #cat GTX108711), and rabbit anti-Iba-1 (1:1,000, GeneTex #cat GTX 100,042). After incubation, tissue sections were treated with the appropriate secondary antibodies anti-rabbit IgG(Vector Laboratories Inc., Burlingame, CA, USA, #cat BA 1000), anti-mouse IgG (Vector Laboratories Inc., Burlingame, CA, USA, #cat BA 2000) and detected with Vectastain ABC (Vector Laboratories Inc.). Next, diaminobenzidine chromogen in 0.1 M tris–HCL buffer was added to immune-reacted sections for a few seconds. Finally, the tissue sections were dehydrated with graded alcohol and mounted with Canada balsam (Kanto Chemical).
5
Cresyl Violet Staining for Gerbil Hippocampus
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Cresyl violet (CV) staining was performed to determine the number of live neuronal cells in the gerbil hippocampus. The brain tissue sections containing the hippocampus were placed on microscopy slides coated with gelatin. After 15 min of staining with CV acetate solution (Sigma‐Aldrich, St. Louis, MO, USA), the slides were rinsed in serial ethanol (70%, 80%, 90%, 95%, 100%) baths for dehydration and Canada balsam (Kanto Chemical, Tokyo, Japan) was used to mount the slides.
6
Histochemical Analysis of Neuronal Damage
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To elucidate the neuronal death/damage induced by MI, we performed CV and F-J B histofluorescence staining as previously described (Lee et al., 2010). In brief, the sections were stained with cresyl violet acetate solution (1.0% (w/v), Sigma-Aldrich, St. Louis, MO, USA), dehydrated in a graded ethanol series, cleared in xylene, and coverslipped. They were then mounted with Canada balsam (Kanto chemical, Tokyo, Japan). For F-J B histofluorescence, the sections were immersed in a F-J B (0.0004%, Histochem, Jefferson, AR, USA) staining solution. After washing, the sections were investigated using an epifluorescent microscope (Carl Zeiss, Göttingen, Germany) with blue (450–490 nm) excitation light and a barrier filter. F-J B-positive cells were counted in a 250 × 250 μm2 square, selected approximately at the center of the cingulate cortex and the piriform cortex. Cell counts were obtained by averaging the total cell numbers from each rat per group.
7
Masson's Trichrome Staining for Myocardial Infarction
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The heart sections were stained to examine the result of MI using Masson's trichrome stain kit (Sigma-Aldrich, St. Louis, MO, USA) according to a method described previously (Ahmet et al., 2013). In brief, the deparaffinized sections were placed in the Biebrich scarlet-acid fuchsin and aniline blue to detect infarction area in the heart, and they were mounted in Canada balsam after the staining (Kanto chemical, Tokyo, Japan). The size of MI was calculated using a previous method (Ahmet et al., 2013) as an average percentage of endocardial and epicardial circumferences in the left ventricle that were identified as infarct area in the Masson's trichrome-stained sections.
8
Organ Fixation and Histological Staining
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Lungs, liver, stomach, and small intestine were perfused with phosphate-buffered saline (PBS) through the right ventricle of the heart, then inflated by injecting 4% paraformaldehyde (PFA)/PBS containing NaN3 (PBS/NaN3) through the trachea, and fixed for 15 min. Then, the whole organs were harvested and kept overnight in 4% PFA/PBS/NaN3 at 4 °C. After fixation, they were dehydrated and embedded in paraffin wax. The paraffin-embedded blocks of the organs were cut into 4 μm tissue sections and stained with a standard HE staining procedure using 1% Eosin Y Solution (Wako) and Mayer’s Hematoxylin Solution (Wako) according to a previously described method37 (link). Then, the specimens were mounted in Canada Balsam (KANTO CHEMICAL CO., INC.) and imaged employing fluorescence microscopy (KEYENCE, BZ-X710).
9
Histological Examination of CA-Induced Liver Damage
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CA-induced pathological alterations in the livers of each group were examined by H&E staining according to our previous study (28 (link)). In brief, the paraffin sections were deparaffinized in xylene and rehydrated in a descending ethanol gradient and washed briefly in distilled water. Next, the sections were stained with hematoxylin solution for 7 min followed by eosin solution for 5 min at room temperature and dehydrated by immersion in a descending ethanol gradient. Finally, they were mounted with Canada balsam (Kanto Chemical Co., Inc.).
CA-induced damage and the hypothermic effect on the CA-induced damage were analyzed under AxioM1 light microscope (magnification, x20; Carl Zeiss AG,) equipped with a digital camera (Axiocam; Carl Zeiss AG) connected to a PC monitor.
CA-induced damage and the hypothermic effect on the CA-induced damage were analyzed under AxioM1 light microscope (magnification, x20; Carl Zeiss AG,) equipped with a digital camera (Axiocam; Carl Zeiss AG) connected to a PC monitor.
10
Hippocampal Cellular Changes Characterization
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To show the morphological evidence of the changes in neurons, astrocytes, and microglia in the hippocampus, immunohistochemical staining was conducted for NeuN, GFAP, and Iba-1, respectively, as previously described [20 (link),57 (link)]. In addition, proliferating cells and neuroblasts were visualized with the immunohistochemistry of Ki67 and DCX. Briefly, animals (n = 5 per group) were anesthetized with a mixture of 75 mg/kg alfaxalone and 10 mg/kg xylazine on the 56th day of diet feeding, and blood was obtained by cardiac puncture in the right ventricle. Thereafter, animals were perfused transcardially, and the brain was coronally sectioned with a 30 μm thickness between 2.0 and 2.7 mm caudal to the bregma based on gerbil stereotaxic coordinates [58 (link)]. Four sections located 150 μm apart were selected and incubated with each antibody; mouse anti-NeuN antibody (1:1000; Merck Millipore, Temecula, CA, USA), rabbit anti-GFAP antibody (1:1000; Merck Millipore), rabbit anti-Iba-1 (1:500; Wako, Osaka, Japan), rabbit anti-Ki67 (1:1000; Abcam, Cambridge, UK), or rabbit anti-DCX (1:2000; Abcam). Immunoreaction was visualized with 3,3′-diaminobenzidine tetrachloride (Sigma, St. Louis, MO, USA) in 0.1 M Tris-HCl buffer (pH 7.2). Sections were dehydrated and mounted on gelatin-coated slides in Canada balsam (Kanto Chemical, Tokyo, Japan).
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