Cells were washed with phosphate-buffered saline (PBS) and fixed with 4% formaldehyde for 10 minutes at room temperature. After washing with PBS, the cells were covered with blocking medium (0.25% Triton X-100 and 5% goat serum in PBS) for 1 hour at room temperature. Then, the cells were incubated with the primary antibodies overnight at 4°C. After washing, the cells were incubated with secondary antibodies for 1 hour at room temperature. 4′,6-diamidino-2-phenylindole (Cat# C0065; Beijing Solarbio Science & Technology, Beijing, China) was used for nuclear staining. Images were captured on a fluorescence microscope (Cat# TH4-200; Olympus, Tokyo, Japan) and analyzed with Image Pro-Plus 6.0 (https://www.mediacy.com/ . Primary antibodies included GPX4 (rabbit; 1:100; Cat# ab125006; Abcam, Cambridge, MA, USA), oligodendrocyte transcription factor 2 (Oligo2; goat; 1:200; Cat# F2418; R&D Systems, Minneapolis, MN, USA) and ferroptosis suppressor protein 1 (FSP1, also known as AIFM2; rabbit; 1:200; Cat# BS7655; Bioss, Woburn, MA, USA). Secondary antibodies were Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) (1:500; Cat# ab150077; Abcam) and Cy3-labeled goat anti-mouse IgG (H+L) (1:500; Cat# A0521; Beyotime Biotechnology, Shanghai, China).
Image pro plus 6
Manufactured by Abcam
Sourced in United Kingdom
Image-Pro Plus 6.0 is a comprehensive image analysis software for visualizing, enhancing, and measuring digital images. It provides a range of tools for processing, analyzing, and quantifying image data from various sources.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg h l, by Abcam (1 mentions)
Cy3 labeled goat anti mouse igg h l, by Beyotime (1 mentions)
Ab15580, by Abcam (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
β actin, by Abcam (1 mentions)
β actin, by Beyotime (1 mentions)
Ab216880, by Abcam (1 mentions)
Ab216327, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Matrigel basement membrane matrix, by BD (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
7 protocols using image pro plus 6
1
Immunofluorescent Staining of Ferroptosis Markers
2
Quantifying Tumor Cell Proliferation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tumor sections were incubated with an anti-Ki-67 antibody (1:500, ab15580, Abcam), after which the percentage of Ki-67-positive cells was quantified using Image-ProPlus 6.0.
3
Western Blotting of Key Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was conducted as in past reports.25 (link),26 (link) Briefly, after proteins were transferred to PVDF membranes, these blots were probed using primary antibodies specific for SOX2 (Seven Hills, OH, USA), β-actin (Beyotime, Nantong, China), LLGL2 (Abcam, MA, USA) or E-cadherin (Cell Signaling Technology, MA, USA). HRP-linked secondary antibodies specific for mouse-IgG (ZSGB-Bio, Beijing, China) or rabbit IgG (Abcam) were then used for protein detection, β-actin was used as a control for normalization, and Image-Pro Plus 6.0 was used for quantification.
4
Investigating Intestinal Permeability via ZSWF
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To investigate the effect of ZSWF on intestinal permeability of DCI mice, Western blot analysis was used to detect the protein expressions of occludin and ZO-1. Proteins from colon tissues were extracted and quantified using the BCA protein quantification kit. Total proteins were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane by wet transfer apparatus. Tight junction protein associated antibodies ZO-1 (ab216880, Abcam, United Kingdom) and occludin (ab216327, Abcam, United Kingdom) were used to incubate the bands overnight at 4°C, where β-actin (13E5, CST, United States) was used as a reference protein, and then, goat anti-rabbit secondary antibodies (ab205718, Abcam, United Kingdom) were used to incubate the bands at room temperature for 2 h. In conclusion, the immunoreactive bands were visualized using enhanced chemiluminescence reagents, and the luminescence intensity was quantitatively analyzed using Image-pro Plus6.0.
5
Renal Histopathology and Fibrosis Assessment
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Renal arteries and kidneys were fixed with 4% paraformaldehyde, dehydrated, embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin-eosin (HE) and periodic acid-Schiff (PAS). Renal nerves received HE staining and immunohistochemical staining for tyrosine hydroxylase (TH, Abcam, Cambridge, UK).
Renal fibrosis was evaluated as described.[34] (link) The percentage of the fibrous area was calculated by Image-Pro Plus 6.0. The intensity and distribution of TH staining was assessed by a semiquantitative scoring system: 0 = no reaction, 1 = patch/very weak reaction, 2 = weak to moderate reaction, and 3 = strong reaction. The renovascular damage was also scored by an ordinal semiquantitative grading system: 0 = no injury (no endothelial loss or medial change), 1 = minimal injury (< 25% vessel circumference in endothelial loss and < 25% of media thickness in depth in medial change), 2 = mild injury (25 to <50% vessel change), 3 = moderate injury (50 to < 75% vessel circumference in endothelial loss and 50 to < 75% of media thickness in depth in medial change), and 4 = severe injury (≥ 75% vessel circumference in endothelial loss and ≥ 75% of media thickness in depth in medial change or injured media thickness < 50% of unaffected media).[35] (link)
Renal fibrosis was evaluated as described.[34] (link) The percentage of the fibrous area was calculated by Image-Pro Plus 6.0. The intensity and distribution of TH staining was assessed by a semiquantitative scoring system: 0 = no reaction, 1 = patch/very weak reaction, 2 = weak to moderate reaction, and 3 = strong reaction. The renovascular damage was also scored by an ordinal semiquantitative grading system: 0 = no injury (no endothelial loss or medial change), 1 = minimal injury (< 25% vessel circumference in endothelial loss and < 25% of media thickness in depth in medial change), 2 = mild injury (25 to <50% vessel change), 3 = moderate injury (50 to < 75% vessel circumference in endothelial loss and 50 to < 75% of media thickness in depth in medial change), and 4 = severe injury (≥ 75% vessel circumference in endothelial loss and ≥ 75% of media thickness in depth in medial change or injured media thickness < 50% of unaffected media).[35] (link)
6
Three-dimensional Mouse Aortic Ring Angiogenesis Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Three-dimensional ex vivo mouse aortic ring angiogenesis assay allows analysis of cellular proliferation, migration, tube formation, microvessel branching, perivascular recruitment and remodeling, providing a more complete picture of angiogenic processes compared with traditional in vitro tube formation assays. We performed the aortic ring assay as previously described [26 (link)], with minor modifications. Briefly, the rings from the aortas of 4 weeks old C57BL/6 mice were inserted between two layers of matrigel basement membrane matrix (BD Biosciences) and cultured in EGM-2 in the presence of DMEM or supernatants from PVAT cultures for 7 days. Endothelial sprouts were stained with antibody to CD31 (abcam) by immunofluorescence and quantitative analysis was performed with software Image-Pro Plus 6.0. Three separate aortic sections were quantified for each aorta, and the results were averaged for each animal.
7
Adipocyte Morphology and UCP1 Evaluation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Hematoxylin and eosin (H&E) staining, Oil Red O staining, and NAFLD activity score assessment were performed according to the published methods.(14 , 19 ) For morphometric analysis of H&E‐stained iWAT and eWAT sections, adipocyte number/per field and the mean adipocyte area were calculated from cross‐sectional areas obtained by using Image‐Pro Plus 6.0 (Media Cybernetics, Silver Spring, MD) and was expressed in 1 × 103 μm.(2 , 20 ) Immunohistochemical staining for uncoupling protein 1 (UCP1) (1:500; Abcam, Cambridge, United Kingdom) was performed in iWAT and eWAT slides.(12 ) The protein expression was semi‐quantified by Image‐Pro Plus 6.0 and expressed as density mean derived from density sum/area sum.(21 )
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!