Western blot analysis was performed for tight junction proteins (zonula occludens-1 (ZO-1), Occludin and Claudin-5) and glial fibrillary acidic protein (GFAP) from amygdala brain tissue homogenates. Lysates were made using RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0), centrifuged at 16,100 × G for 30-min and total protein levels were estimated from supernatant using a BCA kit (23225, Thermofisher). Western blot samples were made using XT sample buffer (1610791, Biorad) with DTT and boiled at 95℃ for 10 min. Samples were resolved in duplicate 4–12% BIS–TRIS gels (3,450,125, Biorad) under reducing condition and transferred to PVDF membrane. Probing was done against ZO-1 (1:1000; ZO1-1A12, Thermofisher), Occludin (1:1000; OC-3F10, Thermofisher), Claudin-5 (4C3C2, Thermofisher), GFAP (1:1000; G3893, Sigma) and beta-actin (1:5000; 8H10D10, Cell Signaling). Signals were detected using chemiluminescence substrate (34075, Thermofisher) with anti-mouse IgG-HRP (1:10,000; GENA93, Millipore Sigma), anti-rabbit IgG-HRP (1:20,000; GENA934, Millipore Sigma) or with fluorescence signals using IRDye 68RD goat anti-mouse (1: 10,000; 926–68070, Li-Cor) and IRDye 800 CW goat anti-rabbit (1:10,000; 926–32211, Li-Cor). Protein levels were quantified by densitometric analysis using ImageJ software.
Oc 3f10
Manufactured by Thermo Fisher Scientific
Sourced in United States
The OC-3F10 is a lab equipment product from Thermo Fisher Scientific. It is a centrifuge designed for general laboratory applications. The OC-3F10 offers a compact and efficient solution for sample separation and preparation tasks.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Zo1 1a12, by Thermo Fisher Scientific (2 mentions)
Epr2489, by Abcam (2 mentions)
Irdye 800cw goat anti rabbit, by LI COR (1 mentions)
Bis tris gel, by Bio-Rad (1 mentions)
Xt sample buffer, by Bio-Rad (1 mentions)
G3893, by Merck Group (1 mentions)
Ab150075, by Abcam (1 mentions)
Ab150113, by Abcam (1 mentions)
A30106, by Thermo Fisher Scientific (1 mentions)
Sp8 confocal microscope, by Leica camera (1 mentions)
A22287, by Thermo Fisher Scientific (1 mentions)
Ab7817, by Abcam (1 mentions)
Alexa 488 or 594 conjugated antibodies, by Thermo Fisher Scientific (1 mentions)
Ab85163, by Abcam (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
Claudin 5, by Thermo Fisher Scientific (1 mentions)
Aer002, by Alomone (1 mentions)
Lamin a c, by Cell Signaling Technology (1 mentions)
13 protocols using oc 3f10
1
Quantification of Tight Junction Proteins in Brain
2
Immunofluorescence Staining of Tight Junction Proteins
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Anti-ZO-1 antibody (ZO1-1A12, ThermoFisher, 1:200 dilution), anti-CD31 (GB13248, ServiceBio, 1:3000 dilution), anti-Occludin (OC–3F10, ThermoFisher, 1:500 dilution), anti-Claudin-5 (Ab-AF0130, Affinity, 1:300 dilution), anti-Tricellulin (48–8400, ThermoFisher, 1:100 dilution), anti-VE-cadherin (36–1900, ThermoFisher, 1:50 dilution) and phalloidin labeled F-actin (A30106, ThermoFisher, 1:400 dilution) were used for immune-fluorescent staining, the second antibodies used were goat anti-mouse IgG H&L (ab150113, Abcam, Alexa Fluor® 488, 1:200 dilution) and donkey anti-rabbit IgG H&L (ab150075, Abcam, Alexa Fluor® 647, 1:200 dilution). Paraffin sections were sealed with 1% bovine serum albumin (Solarbio, CAS:A8010) for 1 h. The first antibody was incubated at 4 °C for 12 h. The second antibody, DAPI (Solarbio, CAS: 28718-90-3) and phalloidin labeled F-actin were incubated at room temperature for 1 h. After each incubation, wash with PBS (Servicebio, lot: G0002-2L) for 3 times, each time for 10 min. For immuno-fluorescent observation, Leica SP8 confocal microscope was applied. The captured data are imaged by LAS_X_2.0.2_15022, and then ImageJ is applied for fluorescence statistics.
3
Cochlear Whole-Mount Immunostaining in Mice
4
Immunohistochemical Analysis of Lrp2 Expression
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Adults (n = 5 for each genotype) were anesthetized and perfused transcardially with 4% PFA in 0.12 M phosphate buffer, pH 7.4. After perfusion, eyes were removed from the skull and postfixed overnight in fresh fixative. Serial frozen sections were processed for immunocytochemistry using sheep anti-Lrp2 (1/2000) and mouse anti-alpha smooth muscle actin (1/250; ab7817, Abcam), anti-occludin (1/100; OC-3F10, ThermoFisher). Alexa 488-or 594-conjugated antibodies (1:200, Invitrogen) were used for secondary detection. Nuclear
5
Western Blot Analysis of Senescence and Membrane Markers
6
Immunofluorescence Staining of Tight Junctions
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Immunofluorescence staining was performed [43 (link)] to evaluate ZO-1 and occludin expression in the colon. Anti-TSLP (rabbit, 1:50; GTX85059, GeneTex, Irvine, CA, USA), anti-IL-6R (rabbit, 1:1000; Invitrogen, Waltham, MA, USA), anti-ZO-1 (rabbit, 1:500; 61–7300, Invitrogen, Waltham, MA, USA), and anti-occludin (mouse, 1:200; OC3F10, Invitrogen, Waltham, MA, USA) antibodies were diluted in 1× PBST supplemented with 0.3% BSA. The slides were wrapped in an aluminum foil to block light and stored at 4 °C for 72 h. Tissue sections were incubated for 1 h with a mixture of Alexa 488-conjugated donkey anti-mouse secondary antibody (1:1000; A21206, Invitrogen, Waltham, MA, USA) or Alexa 594-conjugated donkey anti-mouse secondary antibody (1:1000; A21203, Invitrogen, Waltham, MA, USA).
7
Quantifying ACE2 and Tight Junctions
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The expression of ACE2 in LAD2 was determined by immunostaining with PE-labeled rabbit anti-ACE2 (Bioss, bs-1004R) and detecting with flow cytometry (BD Accuri C6). For detecting tight junction proteins ZO-1, Occludin, Claudin-5 and JAM2 in A549 cells, cells were blocked with 5% BSA in PBS for 1 h at room temperature then incubated with primary antibodies for 2 h at 4 °C. Primary antibodies against ZO-1 (Invitrogen, 402200), Occludin (Invitrogen, OC-3F10), Claudin-5 (Invitrogen, 4C3C2) and JAM-2 (Abcam, EPR2489), were used. A permeabilizing agent (1% FBS and 0.2% Triton X-100 in PBS) was used for ZO-1 intracellular staining. Cells were washed with FACS buffer and then incubated with Alexa Flour 488-labeled goat anti-rabbit or goat anti-mouse IgG (Invitrogen, A11034; Invitrogen, A11001) for 1 h at 4 °C, then cells were analyzed with flow cytometry (BD Accuri C6).
8
Immunohistochemical and Flow Cytometric Analysis of Brain Markers
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For monocyte/macrophages expression anti-Ly6C (1:200 Abcam) was used and followed by anti-rat Alexa 488 (1:1000, Invitrogen). To visualise microglia/macrophages, resident microglia, claudin-5 and ZO-1, anti-Iba1 (1:1000 WAKO), anti-TMEM119 (1:500, Abcam), anti-claudin-5 (1:500, Abcam) and anti-zo-1 (1:500, ThermoFisher scientific) were used, respectively. All were followed by anti-rabbit Alexa 488 (1:1000, Invitrogen). To visualise CatS, anti-CatS (1:100, Santa Cruz) was used followed by anti-mouse Alexa 488 (1:1000, Invitrogen). For FACS analysis reported below the following antibodies were used: occludin monoclonal antibody OC-3F10 (1:100, Invitrogen) P-glycoprotein monoclonal antibody C213 (1:10, Thermo Fisher Scientific). ICAM-1/VCAM-1, anti-mouse CD54 (1:200, e-BioLegend) and anti-mouse CD106 (1:200, e-BioLegend). Secondary coupled antibody used for occludin and p-Glycoprotein was an anti-mouse Alexa 488 (1:200, Life Technology).
9
Immunostaining of Rat Brain Endothelial Cells
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10
Quantifying Tight Junction Protein Expression in Caco-2 Cells
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Briefly, Caco-2 cells were seeded in 24-well plates (5 x 105 cells/well) in medium containing 5 % FBS. After reaching 100 % confluence, the various forms of Lf (each 5 mg/ml) were added to the cells. After 24 h, the cells were washed twice with PBS and then were fixed for 5 min with methanol at 20 °C. After additional two washes with PBS, the cells were stained with a FITC-conjugated anti-occludin antibody (2.5 µg/ml, OC-3F10, Invitrogen, USA) overnight at 4 °C. A semiquantitative scale from 0 to 3 was used to indicate the absence, low levels, intermediate levels, and high levels of tight junction protein expression, respectively (Fig. 1 ).![]()
Fluorescent intensity corresponding to different levels of occludin expression in Caco-2 cells stained with a FITC-conjugated anti-occludin antibody. The semiquantitative scale used is represented in the images shown, with 0–3 indicating an absence, low levels, intermediate levels, or high levels of tight junction protein expression, respectively
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