Immunofluorescence staining of NP cells and the indicated IVD tissues was performed with anti-cortistatin (diluted 1:150, sc-393108, Santa Cruz Biotechnology), anti-TNFα (diluted 1:100, sc-133192, Santa Cruz Biotechnology), anti-Col 2 (diluted 1:100, sc-52658, Santa Cruz Biotechnology), anti-aggrecan (diluted 1:200, 13880-1-AP, Proteintech), anti-Annexin V (diluted 1:100, ab14196, Abcam), anti-NLRP3 (diluted 1:100, ab4207, Abcam), anti-IL-1β (diluted 1:200, ab9722, Abcam), anti-p65 (diluted 1:150, ab86299, Abcam) and anti-MMP13 (diluted 1:100, sc-515284) antibodies. The procedure was conducted as we described previously 35 (link), and images were taken with a fluorescence microscope (Olympus IX51, Japan). The immunofluorescence signal intensities were quantified with ImageJ software 41 (link).
Ab14196
Manufactured by Abcam
Sourced in United Kingdom
Ab14196 is a laboratory equipment product manufactured by Abcam. It serves as a core function without further interpretation or extrapolation on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Ab4207, by Abcam (1 mentions)
Anti tnf α, by Santa Cruz Biotechnology (1 mentions)
Sc 133192, by Santa Cruz Biotechnology (1 mentions)
Sc 52658, by Santa Cruz Biotechnology (1 mentions)
Ab9722, by Abcam (1 mentions)
Ab86299, by Abcam (1 mentions)
Paraffin embedded tissue section protocol, by Promega (1 mentions)
Ab178847, by Abcam (1 mentions)
Micron 4 fundus camera, by Phoenix Pharmaceuticals (1 mentions)
Ab17133, by Abcam (1 mentions)
Ab109189, by Abcam (1 mentions)
Ab702, by Abcam (1 mentions)
Ab16667, by Abcam (1 mentions)
Protein block, by Agilent Technologies (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Ab56301, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
Ab65869, by Abcam (1 mentions)
14 protocols using ab14196
1
Immunofluorescence Analysis of IVD Tissues
2
Apoptosis Detection in Testicular Tissue
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Apoptosis detection in the testes using the DeadEnd Fluorometric TUNEL System was carried out according to the standard paraffin-embedded tissue section protocol (Promega).
For annexin V staining of apoptotic cells, the sections were deparaffinized and rehydrated, followed by antigen retrieval in 10 mM of the sodium citrate buffer. Then, sections were blocked with goat serum in 0.3% Triton X-100 and incubated with primary antibodies of annexin V (Abcam, ab14196, 1:50).
For annexin V staining of apoptotic cells, the sections were deparaffinized and rehydrated, followed by antigen retrieval in 10 mM of the sodium citrate buffer. Then, sections were blocked with goat serum in 0.3% Triton X-100 and incubated with primary antibodies of annexin V (Abcam, ab14196, 1:50).
3
Neuroprotective Effects of Intravitreal Ngb
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To determine the effects of intravitreal Ngb injection on post hypoxia pro-inflammatory and chemotactic chemokines (TNFα, MCP-1, IL-6, IL-1B, RANTES, INFγ, IL-15, and VEGF), 12 rats received the intravitreal injection of 5 ul of Neuroglobin protein (1 mg/ml, ProSpec-Tany Technogene Ltd, CYT-450) in the right eyes and BSS in the left eyes on day 0 (pre-treatment before hypoxia exposure) and day 3 (post hypoxia exposure). On day 1, these 12 rats were subjected to hypoxia using a previously published method of hypoxia9 ,10 . In brief, the rats were placed in a decompression (hypobaric) chamber (Galaxy® 170 R CO2 incubator, New Brunswick) filled with a gas mixture of 7% oxygen and 93% nitrogen for 2 hours and then allowed to recover under normoxic conditions. The retinae from all 18 rats, 36 globes were harvested for analysis of pro-inflammatory cytokines and VEGF (Rat Inflammation and Oxidative Stress ELISA Strip, Signosis, EA-1201 and EA-1501), Ngb quantification by ELISA kit (Rat Neuroglobin ELISA Kit, MyBiosource, MBS704958) and immunohistochemical staining with annexin V (abcam, ab14196, 1:200 dilution), and anti-IBA1 antibodies (abcam, ab178847, 1:200 dilutions) for the detection of activated microglial cells. Activated microglial cells count in the retina was quantified manually from three random sections from each group immunohistochemically stained with anti-IBA1 antibodies.
4
In-vivo Analysis of Retinal Degeneration
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On days 7 and 30, fundus photography and OCT images were taken using micron IV fundus camera (Phoenix Research Laboratories, USA) for both Ngb and sham treated eyes. Clinical signs of vitritis, retinitis and optic neuropathy were examined by an experienced clinician. OCT images were analyzed using InSight software V. 1.1 to determine retinal thickness and sign of retinal degeneration. Electroretinogram (ERG) was recorded after overnight dark-adaptation (>12 hours) using corneal monopolar electrodes and an Espion system (Espion, Diagnosys LLC, USA). The stimulus intensity ranging from −3.0 to 1.0 log cd.s.m−2 was used. The harvested retinae were used to analyze the expression of the chemokines, and histology. The eyes were fixed in 10% neutral buffered formalin immediately after enucleation and embedded in paraffin blocks for histology. These were sectioned into four-micron sections and stained with Hematoxylin and Eosin staining (H&E) for morphology, retinal thickness and features of inflammation (presence of macrophages and lymphocytes). Annexin V immunohistochemistry (abcam, ab14196, 1:200 dilution), a marker for apoptosis, was performed to detect apoptotic cells.
5
Immunohistochemical Analysis of Organoid Sections
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5 μm thick organoid sections were created from paraffin-embedded constructs, and then deparaffinized for staining. IHC was used to visualize biomarkers cytokeratin 5/6 (CK5/6), calretinin, and thrombomodulin. Blocking was performed by incubation under Dako Protein Block for 15 minutes. Primary antibodies CK5/6 (Abcam, ab17133, raised in mouse) and calretinin (Abcam, ab702, raised in rabbit) or CK5/6 and thrombomodulin (Abcam, ab109189, raised in rabbit) were applied to the sections on the slides at a 1:200 dilution in Dako Antibody Diluent and incubated at room temperature for 1 hour. Next, secondary Alexa Fluor 488 or Alexa Fluor 594 antibodies with appropriate species reactivity were applied to all samples at 1:200 in Dako Antibody Diluent and left at room temperature for 1 hour (anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 594, Life Technologies, Carlsbad, CA, A-11070). Sections were then incubated with Dapi for 5 minutes prior to coverslipping. For Annexin V and Ki67 staining (Abcam, Cambridge, MA, ab14196 and ab16667, respectively) in subsequent biomarker-driven experiments, an identical protocol was employed. Fluorescence images were taken using a Leica DM400B Compound Microscope and overlaid for analysis.
6
Immunohistochemical Analysis of ANGPT2 and Annexin V
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Clinical specimens and xenografted lesions were subjected to immunohistochemistry (IHC) analysis as described previously [15 (link)]. The primary antibodies were anti-ANGPT2 (ab56301, Abcam) and anti-Annexin V antibody (ab14196, Abcam). The secondary antibodies were horseradish peroxidase-conjugated anti-rabbit immunoglobulin-G antibody (ab6721, Abcam).
7
Apoptosis and Proliferation Pathway Imaging
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1:200 Rat anti-Brdu (AbCam, ab6326), 1:200 Rabbit anti-Activated Caspase 3 (BD Biosciences, 559565), 1:200 Mouse anti-Phospho-p44/41 (ERK1/2) (Cell Signaling, Antibody #9101), 1:1000 DAPI, 1:200 Rabbit anti-eGFP antibody (ThermoFisher Scientific, OSE00002W), Rabbit anti-Annexin V ( AbCam, ab14196), Rabbit anti-MIF (AbCam, ab65869), Rabbit anti-DDT (AbCam, ab115785).
8
Western Blotting Analysis of Soft Palate
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Western blotting analysis was carried out as reported previously by our lab (20 (link)). Briefly, after the total protein of soft palate tissues was extracted and separated, the samples were transferred onto a polyvinylidene fluoride (PVDF) membrane. Following washing, the membrane was incubated overnight at 4°C with primary antibodies against cAMP (1:1000, Abcam, ab76238), protein kinase A (PKA) (1:1000, Abcam, ab75991), cAMP-regulated guanine nucleotide exchange factor II (Epac2) (1:1000, Abcam, ab193665), rat sarcoma protein (Ras) (1:1000, Abcam, ab52939), c-Jun N-terminal kinase 1/2 (JNK1/2) (1:1000, Abcam, ab4821), Annexiv V (1:500, Abcam, ab14196), GAPDH (1:1000, Abcam, ab8245), and Tubulin (1:1000, Abcam, ab44928). After washing, the membrane was incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 30 min at room temperature, followed by color development using a mixed solution. The image analyzer quantitative system was used to quantitatively determine the intensity of the target and reference proteins.
9
Annexin V Immunodetection by Western Blot
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Proteins were loaded onto 4–12% Bis–Tris Novex gradient gel, and SDS-PAGE was performed under recommended conditions. After electrophoresis, the proteins were transferred onto nitrocellulose membrane using iBlot gel transfer system (Life Technologies, Darmstadt, Germany). The membrane was stained with rabbit annexin V antibody (1:1000, ab14196, Abcam, Cambridge, UK) and developed using the WesternBreeze® chromogenic Western blot immunodetection kit.
10
Histological Analysis of mdx Mouse Muscle
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After the euthanized mice were skinned, the gastrocnemius muscles and diaphragms were carefully removed for fixation in 4% paraformaldehyde for 24 h. Then, the muscles were embedded in paraffin, sectioned to 4 μm, and stained with Sirius Red to observe muscle fibrosis and hematoxylin and eosin to observe muscle histopathology and central nuclei as features of mdx mouse muscle. Muscle restoration was detected by incubating fixed gastrocnemius sections with myosin heavy chain antibody (#MAB4470, at 1/1000 dilution; R&D Systems, MN, USA), annexin V antibody (ab14196, at 1/500 dilution, Abcam), and fibronectin antibody (ab2413, at 1/200 dilution, Abcam) for 18 h at 4 °C, then with Alexa Fluor® 488 AffiniPure Goat Anti-mouse IgG (H+L) (A10680, Thermo Fisher Scientific, Waltham, MA, USA), Alexa Fluor® 488 AffiniPure Goat Anti-rabbit IgG (H+L) (A27034, Thermo Fisher Scientific), and Alexa Fluor® 594 AffiniPure Goat Anti-rabbit IgG (H+L) (A11037, Thermo Fisher Scientific) secondary antibodies. Sections were counterstained with Hoechst 33342 (H1339, Thermo Fisher Scientific).
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