Somite tissues of ZZ and DZ at E21 and E28 were fixed in 4% paraformaldehyde for 19 h at 4°C, then dehydrated using gradient alcohol and embedded with paraffin. Paraffin-embedded samples were cut into 5-μm somite cross sections. The paraffin sections were placed in an oven at 64°C for 30 min and immediately moved to xylene for dewaxing. The sections were rehydrated in gradient alcohol, and antigen retrieval was performed using citrate antigen retrieval solution. Finally, immunofluorescence staining was performed using IHC kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions. The antibodies used were as follows: anti-MyoD antibody (Cell Signaling Technology, 13812S), anti-Pax7antibody (Abcam, ab199010), Anti-rabbit IgG (H+L), F (ab')2 Fragment (Alexa Fluor® 488 Conjugate) (CellSignaling Technology, #4412), and Anti-mouse IgG (H+L), F (ab')2 Fragment (Alexa Fluor® 555 Conjugate) (CellSignaling Technology, #4409). Immunostaining images were obtained with a fluorescence microscope (Nikon, Tokyo, Japan).
Ihc kit
Manufactured by Abcam
Sourced in United Kingdom
The IHC kit is a laboratory equipment designed for the purpose of immunohistochemistry (IHC) analysis. It provides the necessary reagents and tools to perform this technique, which is used to detect and visualize specific proteins or antigens within tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti pax7antibody, by Abcam (1 mentions)
F ab 2 fragment alexa fluor 488 conjugate, by Cell Signaling Technology (1 mentions)
Anti caspase 3 antibody, by Cell Signaling Technology (1 mentions)
Apoptag peroxidase in situ apoptosis detection kit, by Merck Group (1 mentions)
Wash buffer, by Beyotime (1 mentions)
Antibodies to γ h2ax, by Cell Signaling Technology (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
8 protocols using ihc kit
1
Immunofluorescence Analysis of Somite Tissues
2
Histological Analysis of Mouse Lungs
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Lungs isolated from mice were formalin-fixed (10%), paraffin-embedded and sectioned (4 µm) prior to staining with hematoxylin and eosin (H&E) or for use in immunohistochemical analysis. For IHC, samples were deparaffinized with xylene and were successively washed with a gradient of ethanol washes (100–70%). Antigen retrieval was subsequently performed, samples were incubated in a 100 °C water bath immersed in citrate buffer (50 mM of citric acid, pH 6.0) for 20 min and cooled at room temperature. Slides were stained using an IHC kit (Cat#: ab64264, Abcam, Cambridge, UK) using antibodies detailed in Supplementary Table S3 , diluted in 5% BSA and incubated for 1 h at room temperature. Nuclei were stained with hematoxylin. Samples were analyzed by a trained veterinary pathologist (P.K.) using four random sections/organ/mouse and 10 high-powered fields of view (HP-FOVs)/sections at 400× magnification (n = 9 mice/group).
3
Apoptosis and Caspase 3 Detection in Lung Tissue
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Apoptosis was detected in the lung tissue using a TUNEL assay kit (ApopTag® Peroxidase In Situ Apoptosis Detection Kit; Millipore Corporation, Billerica, MA, USA), according to the manufacturer's instructions. Apoptotic changes were visualized with 3,3-diaminobenzidine (DAB) chromogen and counterstained with Harris hematoxylin before mounting.
Caspase 3 expression was visualized using an IHC kit (Abcam, Cambridge, UK) following the manufacturer's protocol. Anti-Caspase 3 antibody (1:200 dilution; Cell Signaling Technology, Danvers, MA, USA) and goat anti-rabbit immunoglobulin G were used as the primary and secondary antibodies, respectively. The DAB chromogen was counterstained with Harris hematoxylin. Each slide was examined in a blinded manner under a light microscope (Leica). Ten random non-overlapping fields per slide were acquired, and quantitative image analysis was performed using an image analyzer (IMT i-Solution software, Houston, TX, USA).
Caspase 3 expression was visualized using an IHC kit (Abcam, Cambridge, UK) following the manufacturer's protocol. Anti-Caspase 3 antibody (1:200 dilution; Cell Signaling Technology, Danvers, MA, USA) and goat anti-rabbit immunoglobulin G were used as the primary and secondary antibodies, respectively. The DAB chromogen was counterstained with Harris hematoxylin. Each slide was examined in a blinded manner under a light microscope (Leica). Ten random non-overlapping fields per slide were acquired, and quantitative image analysis was performed using an image analyzer (IMT i-Solution software, Houston, TX, USA).
4
Ovarian Vessel Density Measurement
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After deparaffinization and hydration, the ovarian sections were placed in sodium citrate solution and boiled for antigen retrieval. The following process was performed using an IHC kit (Abcam). Briefly, sections were treated with hydrogen peroxide and blocked, followed by incubation with primary antibodies against von Willebrand factor (vWF, newly formed vessels marker, 1:500, Boster), CD34 (endothelial marker, 1:2000, Abcam), and DDX4 (oocyte marker, 1:2000, Abcam). Then, the sections were washed and sequentially incubated with biotinylated anti-mouse/rabbit IgG, streptavidin peroxidase, and DAB chromogen solution, and finally, counterstained with hematoxylin. The negative control samples received identical treatment, except primary antibodies were omitted, and exhibited no specific staining. For quantification, four ovarian sections from each mouse were subjected to IHC, and three randomly selected high-power fields in each section were analyzed. The final data represent the average microvessel density (MVD).
5
Evaluating Mucus and MMP-9 in Lung Tissues
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Lung tissues were processed as described previously (17 (link)). To evaluate mucus production and airway inflammation, lung tissue was stained with periodic acid-Schiff (PAS; IMEB Inc. San Marcos, CA) or hematoxylin and eosin (H&E; BBC Biochemical, Mount Vemon, WA). The score of mucus production was calculated as the percentage of purple (positive) stained cells in the total cell count. MMP-9 protein was stained using an immunohistochemistry (IHC) kit (Abcam) following the manufacturer’s instructions. A primary antibody against MMP-9 (1:300; Cell Signaling Technology, Danvers, MA) was used for IHC staining. Brown stained cells are positive for MMP-9. Quantitative analyses were conducted with a light microscope in a blinded manner (Leica Microsystems) at 10× and 20× objective lenses using the IMT i-Solution software (Vancouver, North Road Burnaby, Canada).
6
Quantifying DNA Damage in Xenograft Tumors
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Samples from xenograft tumors were fixed using formalin and then used for immunohistochemical staining to measure γH2A.X expression. Tissues were dehydrated in graded ethanol solutions, cleared in 3 changes of xylene, and penetrated in heated paraffin (56–58 °C). The tissues were embedded in paraffin, cut into 4 to 6 mm sections, and placed onto slides. Before staining, deparaffinization and rehydration were performed. Antigen retrieval was performed using a pressure cooker. The slides were incubated in 1× target retrieval solution (Beyotime, Haimen, China) at 120 °C for 4 min at 18 to 20 psi. Endogenous hydrogen peroxidase activity was blocked with hydrogen peroxide for 10 min, followed by rinsing with wash buffer (Beyotime, Haimen, China). The slides were incubated with the appropriate antibodies. The antibodies to γH2A.X were purchased from Cell Signaling Technology, Inc (Danvers, MA, USA). The secondary antibodies against mouse or rabbit IgG were supplied in an IHC kit from Abcam (Cambridge, MA, USA).
7
Perfusion-Fixed Brain Tissue Imaging
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The mice were fixed with a perfusion of 4% paraformaldehyde (PFA). Whole brains were subsequently immersed in a 4% PFA solution for 3 days, after which they were moved to a solution of 30% sucrose in phosphate-buffered saline (PBS) for 2 days at 4 °C. To aid in visualizing brain structures, the sections were stained with antibodies recognizing the neuronal marker NeuN. The sections were blocked for 1 h at room temperature in PBS containing 3% bovine serum albumin and 0.3% Triton X-100 before incubation with a 1:1000 dilution of anti-NeuN antibody (Thermo) overnight at 4 °C. The sections were then washed in PBS and treated with streptavidin peroxidase and 3,3′-diaminobenzidine (DAB) using an IHC kit (Abcam). The sections were viewed with an Axio Scan.Z1 slide scanner (Carl Zeiss) using a 20× objective lense, 0.325 × 0.325 × 0.490 μm/pixel resolution, 24-bit pixel depth, and a scan average of 13.
8
Tissue Preparation and Immunofluorescence Staining Protocol
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LDM or forelimbs were fixed in 4% paraformaldehyde for 19 h at 4°C, then dehydrated using gradient alcohol and embedded with paraffin. Paraffin embedded samples were cut into 5-μm sections. The paraffin sections were placed in an oven at 64°C for 30 min and immediately moved to xylene for dewaxing. The sections were rehydrated in gradient alcohol and antigen retrieval was performed using citrate antigen retrieval solution. Finally, immunofluorescence staining was performed using IHC kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions, and immunostaining images were obtained with a fluorescence microscope (Nikon).
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