Female BALB/c wild–type mice were purchased from Japan SLC, Inc. (Hamamatsu, Japan) and housed in sterile conditions. The experiments began when the mice were 8 weeks old. 4T1 tumor cells in culture were harvested and re–suspended in phosphate–buffered saline. Viable 4T1 cells (1 × 105) were injected into the left thoracic mammary gland. Tumor size and body weight were measured every 3 days for 21 days. DFX (160 mg/kg) was suspended in saline and administered orally using a probe 3 days/week. Treatment started 3 days after tumor implantation (n = 10). The control group received saline administered by oral gavage 3 days/week. Tumor volume was calculated using the following formula: Volume = (width)2 × length/2. At the end of the experiment, the animals were sacrificed, and the tumors were collected for weight measurement and histological analysis. Bouin’s solution (FUJIFILM Wako Pure Chemical Corporation) was used to perfuse the lungs, which were then fixed in the same solution. The number of metastatic tumors on the lungs was counted under a stereomicroscope.
Bouin s solution
Manufactured by Fujifilm
Sourced in Japan
Bouin's solution is a fixative used in histological and cytological sample preparation. Its core function is to preserve the cellular structure and morphology of tissue samples for subsequent analysis and observation.
Automatically generated - may contain errors
Lab products found in correlation
Neutral buffered formalin, by Fujifilm (1 mentions)
4t1 cell, by American Type Culture Collection (1 mentions)
Dab solution, by Vector Laboratories (1 mentions)
Triton x 100, by Bio-Rad (1 mentions)
Abc reagent, by Vector Laboratories (1 mentions)
Biotinylated horse anti mouse igg, by Vector Laboratories (1 mentions)
Glutaraldehyde, by Nacalai Tesque (1 mentions)
Formalin, by Fujifilm (1 mentions)
Haematoxylin and eosin, by Muto Pure Chemicals (1 mentions)
Szx12, by Olympus (1 mentions)
Female balb c mice, by Japan SLC (1 mentions)
Balb c nu nu mice, by Japan SLC (1 mentions)
Bz 9000, by Keyence (1 mentions)
7 protocols using bouin s solution
1
Orthotopic 4T1 Mammary Tumor Model
2
Evaluating Anti-GM-CSF Therapy in 4T1 Tumor Model
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Wild type female BALB/c mice were purchased from Japan SLC, Inc. (Hamamatsu, Japan). 4T1 cells (ATCC, Manassas, VA, USA) were cultured in RPMI 1640 supplemented by 10% FBS, 2 mM L-glutamine, penicillin/streptomycin and sodium pyruvate. One million cells were grown to 50 to 80% confluence in T-75 tissue culture flasks. Cells were detached with 0.2% trypsin-EDTA, washed once with medium, three times with PBS and resuspended in PBS at 1 × 106/mL. One hundred μL of cell suspension (1 × 105 cells) were injected into the 3rd mammary pad of female mice. Mice were separated into 2 groups; 5 mice in the control group received i.p. injection of normal rat IgG (100 μg in 100 μL PBS) whereas 5 mice in the experiment group received anti-mouse GM-CSF IgG (100 μg in 100 μL PBS) twice a week, for 2 weeks. Blood was collected by heart puncture, and sera were isolated and stored at −80 °C until use. Tumors were harvested and a half was fixed in 10% neutral buffered formalin (Wako) and the other half was in RNAlater. Lungs were perfused with Bouin’s solution (Wako), fixed in the same solution, and then the number of tumor nodules was counted by eye. Tumor length and width were measured using a caliper and tumor volume was calculated using the following formula: Volume = (width)2 × length/2.
3
Immunostaining of Corneal Oxidative Damage
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The excised corneas were fixed in Bouin’s solution (Wako Pure Chemical Industries, Osaka, Japan) for 1 hour at 4 °C, washed 3 times for 10 minutes each in PBS with 1% Triton X-100 (Bio-Rad Laboratories, CA) (PBST), and treated with acetone for 3 minutes at −20 °C. They were further washed 3 times for 10 minutes with PBST and incubated in 10% horse serum diluted in PBST to block nonspecific staining. The corneas were then incubated in a 1:30 dilution of a monoclonal antibody against 8-OHdG (control n = 5, H2 group n = 6) or 4-hydroxy-2-nonenal (4-HNE) (control n = 3, H2 group n = 5) overnight at 4 °C (18). Both antibodies were purchased from the Japan Institute for the Control of Aging (Shizuoka, Japan). The corneas were washed with PBST 3 times for 10 minutes and incubated with horse biotinylated anti-mouse IgG (Vector Laboratories, Burlingame, CA) for 1 hour at room temperature. Tissues were then washed with PBST 3 times for 10 minutes and incubated with ABC reagent (Vector Laboratories) for 1 hour at room temperature. After being washed with PBST 3 times for 10 minutes, corneas were stained with DAB solution (Vector Laboratories) for 10 minutes at room temperature. Finally, they were washed with distilled water for 5 minutes each and mounted on slides.
4
Tissue Preparation and Staining Techniques
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Whole larvae were fixed overnight in a solution of 4% paraformaldehyde (PFA) and 5%
sucrose in 0.1 M phosphate buffer at 4°C, and embedded in paraffin. Adult and juvenile
zebrafish were euthanized by immersion in ice-cold water or 1:1,000 dilution of
2-phenoxyethanol. After the euthanasia, adult and juvenile zebrafish were decapitated, and
the heads were fixed overnight in Bouin’s solution (Wako Pure Chemical Industries, Ltd.,
Osaka, Japan) at 4°C, and thereafter embedded in paraffin. The paraffin blocks were cut in
5 µm thick sections, and stained with hematoxylin and eosin
(H&E).
The eyes of rats were fixed overnight in a solution of 10% formalin (Wako Pure Chemical
Industries, Ltd.): 25% glutaraldehyde (Nacalai Tesque, Inc., Kyoto, Japan) (9:1) at 4°C,
and thereafter embedded in paraffin. H&E stained specimens were prepared under routine
method.
sucrose in 0.1 M phosphate buffer at 4°C, and embedded in paraffin. Adult and juvenile
zebrafish were euthanized by immersion in ice-cold water or 1:1,000 dilution of
2-phenoxyethanol. After the euthanasia, adult and juvenile zebrafish were decapitated, and
the heads were fixed overnight in Bouin’s solution (Wako Pure Chemical Industries, Ltd.,
Osaka, Japan) at 4°C, and thereafter embedded in paraffin. The paraffin blocks were cut in
5 µm thick sections, and stained with hematoxylin and eosin
(H&E).
The eyes of rats were fixed overnight in a solution of 10% formalin (Wako Pure Chemical
Industries, Ltd.): 25% glutaraldehyde (Nacalai Tesque, Inc., Kyoto, Japan) (9:1) at 4°C,
and thereafter embedded in paraffin. H&E stained specimens were prepared under routine
method.
5
Ovarian Follicle Histomorphometry and Classification
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Histological assessment and classification of follicle structure were conducted as described previously with slight modification [18 (link),21 (link)]. Briefly, at least three pieces of ovarian tissue in each group were fixed with Bouin’s solution (FUJIFILM Wako Pure Chemical Corporation), kept at 4 °C overnight, dehydrated in a series of ethanol solutions (70–100%), and embedded in paraffin. Serial sections (5 μm thick) of ovarian tissues were prepared and stained with haematoxylin and eosin (both Muto Pure Chemicals CO. Tokyo, Japan). To avoid double counting, three sections, each at least 20 µm apart, were assessed by light microscopy (Nikon ECLIPE E600, Nikon).
Follicles within the ovarian tissues were classified as primordial (<80 um), primary (0.08–1 mm), or pre-hierarchical (>1 mm) [8 (link)]. All the follicles were further characterized as follows: ‘normal’, when the layer of granulosa cells is attached to the spherical oocyte surrounding it and the homogenous ooplasm contains a tiny granulated nucleus, or ‘abnormal’, wherein aggregation and shrinkage of nuclear chromatin and wrinkling of the nuclear membrane were regarded as signs of atresia [6 (link)]. The proportion of morphologically normal follicles per section was calculated by dividing the number of normal follicles by the total number of assessed follicles.
Follicles within the ovarian tissues were classified as primordial (<80 um), primary (0.08–1 mm), or pre-hierarchical (>1 mm) [8 (link)]. All the follicles were further characterized as follows: ‘normal’, when the layer of granulosa cells is attached to the spherical oocyte surrounding it and the homogenous ooplasm contains a tiny granulated nucleus, or ‘abnormal’, wherein aggregation and shrinkage of nuclear chromatin and wrinkling of the nuclear membrane were regarded as signs of atresia [6 (link)]. The proportion of morphologically normal follicles per section was calculated by dividing the number of normal follicles by the total number of assessed follicles.
6
Metastatic Tumor Modeling in BALB/c Mice
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Female BALB/c mice were purchased from Japan SLC, Inc. (Hamamatsu, Japan). MD. One million tumor cells were seeded in a T-75 tissue culture flask and grown to 50–80% confluence. Cells were detached with 0.2% trypsin–EDTA, washed once with complete medium and three times with PBS, and resuspended in PBS at 1 × 106 cells/ml. One hundred and thousand cells in 100 μl PBS were injected into the third left mammary pad. Tumor tissues were excised and fixed in 10% formalin. Lungs were perfused with Bouin’s solution (Wako, Osaka, Japan) and fixed in the same solution, and then, the number of tumor nodules was counted by eye. After fixation in Bouin’s solution, subpleural lung surface metastases were easily identified by their light, white appearance. Tumor length and width were measured using a caliper, and tumor volume was calculated using the following formula: Volume = (width)2 × length/2.
LM.4T1-RFP and HM.4T1-GFP cells were implanted into nude (BALB/c-nu/nu) mice (Japan SLC, Inc.). Four weeks later, lungs were removed and metastases foci of GFP and RFP expression cells were immediately visualized using a fluorescence microscope (Olympus stereoscopic microscope, SZX12). Lungs were then fixed in Bouin's solution to detect tumor nodules. LM-Wnt7a KO, HM-Wnt7a KO, and LM-Rab27a KO cells were also implanted into the mammary pad of BALB/c-nu/nu mice.
LM.4T1-RFP and HM.4T1-GFP cells were implanted into nude (BALB/c-nu/nu) mice (Japan SLC, Inc.). Four weeks later, lungs were removed and metastases foci of GFP and RFP expression cells were immediately visualized using a fluorescence microscope (Olympus stereoscopic microscope, SZX12). Lungs were then fixed in Bouin's solution to detect tumor nodules. LM-Wnt7a KO, HM-Wnt7a KO, and LM-Rab27a KO cells were also implanted into the mammary pad of BALB/c-nu/nu mice.
7
Histological Analysis of Testes
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Testes were fixed with Bouin’s solution (FUJIFILM Wako Pure Chemical) at 4°C overnight with gentle shaking. Fixed testes were washed in PBS and then dehydrated by serial incubations in
ethanol (70–100%, v/v). Dehydrated testes were embedded in paraffin, sectioned, or rehydrated as described under Immunohistochemistry. For histological analysis, sections were stained with
hematoxylin and eosin (HE) by a standard HE staining procedure. Stained sections were dehydrated by serial incubations in ethanol (50–100%, v/v) followed by permeation by incubation twice in
Lemosol A (FUJIFILM Wako Pure Chemical). Sections were mounted with a coverslip with Mount-Quick (Daido Sangyo, Saitama, Japan) and observed under a fluorescence microscope (BZ-9000,
Keyence).
ethanol (70–100%, v/v). Dehydrated testes were embedded in paraffin, sectioned, or rehydrated as described under Immunohistochemistry. For histological analysis, sections were stained with
hematoxylin and eosin (HE) by a standard HE staining procedure. Stained sections were dehydrated by serial incubations in ethanol (50–100%, v/v) followed by permeation by incubation twice in
Lemosol A (FUJIFILM Wako Pure Chemical). Sections were mounted with a coverslip with Mount-Quick (Daido Sangyo, Saitama, Japan) and observed under a fluorescence microscope (BZ-9000,
Keyence).
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