HeLa cells, grown on borosilicate cover glasses (Thermo Fisher Scientific), were co‐transfected with mCherry‐Parkin (Addgene, 23956) and either mitochondria‐targeted GFP or one of the GFP‐tagged IF1 clones (wild type, dominant negative E30A, or constitutively active H49P) as previously demonstrated (Faccenda et al., 2017 (link)). After 48 h from transfection, cells were fixed with 4% paraformaldehyde in PBS (10 min) and prepared for immunostaining. Briefly, cells were permeabilized in 0.1% Triton X‐100 in PBS for 15 min and incubated in blocking solution (10% normal goat serum, Thermo Fisher Scientific, PCN5000; 3% BSA, Sigma‐Aldrich, A2153; 0.01% Triton X‐100 in PBS) for 1 h. Cells were then incubated overnight with an anti‐ATPβ (Abcam, ab14730) 1:500 in blocking solution, washed 5 times in PBS, and incubated for 2 h with an Alexa Fluor 647‐conjugated anti‐Mouse IgG1 (Thermo Fisher Scientific, A21240) 1:500 in blocking solution. After a further washing (5 times in PBS), cells were mounted on slides using mounting medium with DAPI (Abcam, ab104139).
Pcn5000
Manufactured by Merck Group
The PCN5000 is a laboratory instrument designed for polymerase chain reaction (PCR) amplification of nucleic acid samples. The device features a temperature-controlled thermal block for precise temperature regulation during the PCR thermal cycling process. The PCN5000 provides the core functionality required for PCR-based analysis and testing in a research or diagnostic laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Ab14730, by Abcam (1 mentions)
Ab104139, by Abcam (1 mentions)
A2153, by Merck Group (1 mentions)
Mcherry parkin, by Addgene (1 mentions)
S8032, by Merck Group (1 mentions)
T8787, by Merck Group (1 mentions)
Ab13970, by Merck Group (1 mentions)
B 1355, by Vector Laboratories (1 mentions)
A11039, by Thermo Fisher Scientific (1 mentions)
D9663, by Merck Group (1 mentions)
Cy3 streptavidin, by Jackson ImmunoResearch (1 mentions)
C8035, by Merck Group (1 mentions)
Sp 2002, by Vector Laboratories (1 mentions)
3 protocols using pcn5000
1
Mitochondrial Dynamics Visualized in HeLa Cells
2
High-Throughput Cell Staining Protocol
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This protocol was used for staining cells in a 96-well (VWR #82050-748) format and a shaker was used during antibody incubations to obtain uniform staining. The cells were washed twice with 1x PBS (100 µl) and fixed with 75 µl of 4% paraformaldehyde (VWR #AA43368-9M) per well (15 min at RT). Cells were washed with PBS and 75 µl of Blocking Buffer (5% Goat Serum Life Technologies #PCN5000 with 0.32% Triton X-100 Sigma #T8787 in 1× PBS) added per well (1 h at RT). After PBS wash, 50 μl/well of primary antibody (at recommended dilutions in Ab protocol) in the Antibody Buffer (1% BSA and 0.3% Triton X-100 in 1x PBS) was added and incubated at RT, shaking gently in an orbital shaker for 1–2 h for same day processing or overnight at 4 °C while shaking. Cells were washed with PBS and 50 μl/well of secondary antibody in Antibody Buffer (at recommended dilutions in Ab protocol) was added with incubation at RT, shaking gently in an orbital shaker for 1–2 h. After final wash in 1x PBS, plates were wrapped in aluminum foil and stored at 4 °C with 100 µl 0.05% Sodium Azide (NaN3, Sigma #S8032) in PBS.
3
Immunohistochemical Visualization of Glycoconjugates
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Coronal sections were washed three times for 10 minutes in PBST. For wisteria floribunda agglutinin (WFA) staining, samples were first blocked using a biotin blocking kit (Vector Labs SP2002). Sections were then incubated in block (PBST +5% Normal Goat Serum (NGS)(Thermo PCN5000), +5% Normal Donkey Serum (NDS)(Sigma D9663)) for 1 hour and then incubated in primary antibody solution(PBST + 2.5% NGS and 2.5% NDS) overnight at 4 °C. Primary antibodies used were chicken anti-GFP (1:500 Abcam ab13970), mouse anti-CS-56 (1:200 Sigma C8035), biotinylated WFA (1:1000 Vector Labs B-1355). Sections were washed three times for 10 minutes in PBS and then incubated for 1 hour at RT in secondary antibody solution (0.5x blockPBST +2.5% NGS and 2.5% NDS). Secondary antibodies used were goat anti-chicken Alexa 488 (1:1000 Invitrogen A11039), 10 μM HTL646 (1:500), Streptavidin-cy3 (1:1000 Jackson Immuno Research 016-160-084) and Hoescht 33343 (1:10K Invitrogen H3570). sections were washed three times for 10 minutes in PBS, and then placed on slides. Sections were mounted with flouromount gold and coverslips (TedPella 260152) and left to dry overnight at RT and stored at −20 °C.
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