Paraffin-embedded organotypic culture slides were deparaffinized and rehydrated following standard protocols. Antigen retrieval was performed by boiling the slides in 1M Tris EDTA, pH8.0 for 10 min. Slides were allowed to cool down to room temperature before incubation in 10% normal goat serum blocking solution (Thermo Fisher Scientific) for 30 min. Primary antibodies of anti-APE1 (diluted at 1:500) and anti-MMP-14 (diluted at 1:200) were added to the slides and incubated overnight at 4°C in a humidified chamber. The next day following incubation, the slides were washed with PBS and incubated with Alexa Fluor conjugated anti-mouse or anti-rabbit secondary antibody (Fluor-488 or Fluor-586) for 1 h at room temperature, protected from light. The slides were washed again with PBS and mounted with a Vectashield mounting medium with DAPI (Vector Laboratories). Images were captured by All-in-One Fluorescence Microscope (BZ-X700, Keyence Corp, Atlanta, GA, USA).
Normal goat serum blocking solution
Manufactured by Thermo Fisher Scientific
Normal goat serum blocking solution is a laboratory reagent used to block non-specific binding in various immunoassay techniques, such as Western blotting, immunohistochemistry, and ELISA. It is a sterile-filtered, ready-to-use solution containing normal goat serum that can effectively minimize background staining and improve the specificity of antibody-based detection methods.
Automatically generated - may contain errors
Lab products found in correlation
All in one fluorescence microscope, by Keyence (1 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (1 mentions)
Ab104139, by Abcam (1 mentions)
Bz x710 all in one fluorescence microscope, by Keyence (1 mentions)
Fv1000 confocal microscope, by Olympus (1 mentions)
Hardset antifade mounting medium, by Vector Laboratories (1 mentions)
Alexa fluor 488 goat anti rat igg h l, by Thermo Fisher Scientific (1 mentions)
5 protocols using normal goat serum blocking solution
1
Immunostaining of Paraffin-embedded Tissues
2
Immunofluorescence Staining of Paraffin Sections
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Paraffin-embedded sphere slides were deparaffinized and rehydrated following standard protocols. Antigen retrieval was performed by boiling the slides in 1 mmol/L Tris EDTA, pH 8.0 for 20 min. Slides were allowed to cool down to room temperature before incubation in 10% normal goat serum blocking solution (Thermo Fisher Scientific) for 1 h. Slides were incubated with primary antibodies overnight at 4 °C in a humidified chamber. Slides were washed with PBS and incubated with secondary antibody (Alexa Fluor 488 and Alexa Fluor 568) for 1 h at room temperature. Finally, slides were washed and mounted using mounting medium with DAPI (Abcam; ab104139). Fluorescent signals were captured using BZ-X710 KEYENCE All-in-One Fluorescence Microscope.
3
Visualizing CRT Surface Localization
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Immunofluorescence was performed to visualize CRT surface localization. Neuro-2a and 9464D cells (1×104 cells) were seeded in 4-well chamber slides (Lab-Tek). NB cells were treated with vehicle or STING-NPs for 24 hours, fixed with 4% paraformaldehyde solution for 20 min, incubated with PBS (0.5% Triton-X) for 15 min, and blocked using 10% normal goat serum blocking solution (Thermo Fisher Scientific, Waltham, Massachusetts) for 1 hour at room temperature. Cells were stained with an Alexa Fluor 647-conjugated anti-CRT antibody (ab196159, 1/200, Abcam) overnight at 4°C, washed three times, followed by staining with 5 µg/mL Alexa Fluor 488-conjugated wheat germ agglutinin for 30 mins to visualize the cell surface membrane. Slides were washed three times and mounted with DAPI nuclear dye and visualised under an Olympus FV-1000 confocal microscope. High magnification images were obtained under the 40× objective lens.
4
Quantifying Autophagy Flux in Cardiomyocytes
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The autophagy flux was measured as described previously2 (link), 21 (link). In brief, the NRCs were plated on coverslips and infected with an mRFP-GFP-LC3 lentivirus (Hanbio. Co. LTD, Shanghai, China) at a multiplicity of infection of 8. The NRCs were exposed to the virus overnight, after which media were aspirated and fresh media were applied. Then the NRCs were subjected to different treatments and fixed with paraformaldehyde in phosphate-buffered saline (PBS). After rinsing with PBS, the cells were permeabilized with 0.3% Triton X-100 in 10% normal goat serum blocking solution (Invitrogen, Life Technologies) for 60 min. Coverslips were mounted on slides with Hardset anti-fade mounting medium (Vector Laboratories), and confocal imaging was performed as described above (mRFP 594 nm ex., 667 nm em.; GFP 488 nm ex., 543 nm em.). The puncta of six cells in each experimental group were counted after obtaining digital images. The number of yellow puncta in the merged channel represented the number of autophagosomes. The number of autolysosomes (as a result of autophagosome-lysosome fusion) was represented by the number of red puncta.
5
Immunostaining of Insulin in MIN6 and MS1 Cells
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MIN6 + MS1 cell-loaded PMs cultured for
7 days were first recovered and fixed using 4% PFA for 30 min at room
temperature followed by extensive washing. Subsequently, the samples
were immersed in 0.5% Triton X-100/PBS at 4 °C for 15 min and
blocked with a 10% normal goat serum-blocking solution (Invitrogen).
Insulin was stained by exposure to an anti-insulin primary antibody
(1:100 dilution, R&D Systems) at 4 °C overnight, followed
by incubation with secondary antibody Alexa Fluor 488 goat anti-rat
IgG (H + L) (1:100 dilution, Invitrogen) for 2 h at room temperature.
A DAPI mounting medium was used before the samples were observed under
CLSM.
7 days were first recovered and fixed using 4% PFA for 30 min at room
temperature followed by extensive washing. Subsequently, the samples
were immersed in 0.5% Triton X-100/PBS at 4 °C for 15 min and
blocked with a 10% normal goat serum-blocking solution (Invitrogen).
Insulin was stained by exposure to an anti-insulin primary antibody
(1:100 dilution, R&D Systems) at 4 °C overnight, followed
by incubation with secondary antibody Alexa Fluor 488 goat anti-rat
IgG (H + L) (1:100 dilution, Invitrogen) for 2 h at room temperature.
A DAPI mounting medium was used before the samples were observed under
CLSM.
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