IEM analysis was performed at the electron microscopy core facility on campus. Briefly, cells were fixed for 1 h, followed by washing with 0.1 M PB buffer. Samples were then dehydrated with ethanol, embedded, cut into 80 nm-thick slides by Leice EM UC6, and then mounted on formvar/carbon-coated 200 mesh gold grids. Then slides were incubated with antibodies (p62 Sigma-Aldrich 1:20, P0067; FTO abcam, ab92821, 1:20) in 1% BSA. After rehydration with PBS and blocking with 1% BSA, slides were next washed with PBS, followed by blocking with 0.5% BSA again, and then incubated with gold-conjugated secondary antibodies (10 nm for FTO or 15 nm for p62, 1:10) in 0.5% BSA. After washing, samples were stained briefly with Uranyl Acetate and Lead Citrate, air dried, and then examined under 300KV at FEI Tecnai F30.
P0067
Manufactured by Abcam
P0067 is a primary antibody that recognizes the Histone H3 (tri methyl K9) protein. This antibody is designed for use in immunohistochemistry and western blotting applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab92821, by Abcam (2 mentions)
Tecnai f30, by Thermo Fisher Scientific (1 mentions)
Ab51031, by Abcam (1 mentions)
Ab61179, by Abcam (1 mentions)
L8918, by Merck Group (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (1 mentions)
Ab192890, by Abcam (1 mentions)
Duolink detection kit, by Merck Group (1 mentions)
Ab48394, by Abcam (1 mentions)
4 protocols using p0067
1
Immunoelectron Microscopy Analysis of Cellular Proteins
2
Western Blot Analysis of Autophagic Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Macrophage supernatants were removed post treatment and the cells were washed twice with phosphate buffered saline (PBS). The cells were lysed in lysis buffer and primed with a protease inhibitor (Roche Applied Science, 10-519-978-001). Then, 30 ug of protein was separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were immunoblotted for LC3 (Sigma-Aldrich, L8918), p62 (Sigma-Aldrich, P0067), beclin-1 (Abcam, ab51031), actin (Abcam, ab8226-100), IFNGR1 (Abcam, ab61179), IFNGRbeta (Abcam, ab84524), and calreticulin (Stressgen, #SPA-600). Protein bands were detected with HRP-conjugated secondary antibodies visualized using enhanced chemiluminescent (ECL) reagents (Life Sciences, RPN2106).
3
Quantitative Analysis of Autophagy Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PLA assay was performed according to the manufacturer’s instructions (Sigma). Briefly, cells were first fixed with 4% paraformaldehyde/PBS and then permeabilized in 0.5%(v/v) Triton X-100. After blocking with 5% normal goat serum (Invitrogen, Carlsbad, California), cells were incubated overnight with the following primary antibodies at 4 °C in 1XPBS/1%BSA: anti-p62 (Rabbit) (Sigma-Aldrich, P0067, 1:400); anti-LC3B (rabbit) (Abcam, ab192890,1:200), and anti-FTO (mouse) (Abcam, ab92821, 1:200). In-situ PLA detection was performed using a Duolink Detection Kit (Sigma-Aldrich) with a pair of nucleotide-labeled secondary antibodies. After ligation and amplification of PLA probes, signals were examined using a fluorescence microscope (Olympus IX71).
4
Histopathological analysis of Labrador Retriever dogs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Autopsy was performed in 8 LR dogs (dog Nos. 1-8). Samples of the major internal organs, skin, central nervous system (CNS), and peripheral nerves were formalin fixed, paraffin embedded, and processed for routine histology. The brain and a skin biopsy were formalin fixed and examined from the ninth euthanized dog (dog No. 9). Skin biopsies from the dorsal flank area of 4 additional living LRs were formalin fixed, paraffin embedded, and processed for routine histology. Tissue sections of the formalin-fixed material were stained with hematoxylineosin (HE), periodic acid-Schiff (PAS), and PAS-diastase. Fresh-frozen tissue samples were prepared from the pancreas and skin of 2 affected (dog Nos. 2 and 3) and 1 control dog (control dog No. 1) and stained with PAS and Oil-red-O for glycoproteins and lipids, respectively. Antibodies used for immunohistochemical staining included anti-p62/sequestosome 1 as marker for autophagic cargo (Sigma-Aldrich P0067), anti-LC3A/B for autophagosomal membranes (LC3B; Abcam ab48394), and anti-LAMP2 for the integral membrane protein
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!