Formalin-fixed, paraffin-embedded sections were deparaffinized, and heat-induced antigen retrieval was performed with Tris–EDTA buffer (pH 9.0). This process was not performed on free-floating mouse brain sections. After blocking with 10% goat or donkey serum and 0.02% Triton-X 100 in PBS for 1 h at RT, sections were incubated overnight at 4°C with primary antibodies (listed in Additional file 5 : Table S2) (when MJFR-14–6-4–2 antibody was used, 0.5 M NaCl was added to the buffer). These sections were incubated with goat- or donkey-derived secondary antibodies (Alexa Fluor 488, 594, and 647, 1:400, Thermo Fisher) for 2 h at RT. The sections were incubated with Vector TrueVIEW Autofluorescence Quenching Kit (Vector Laboratories) for 5 min to reduce autofluorescence, covered with Vibrance Antifade Mounting Medium with DAPI (Vector Laboratories), and observed with an FV1000 confocal microscope (OLYMPUS).
Vibrance antifade mounting medium with dapi
Manufactured by Vector Laboratories
Sourced in United States
Vibrance Antifade Mounting Medium with DAPI is a ready-to-use, glycerol-based aqueous mounting medium designed to preserve fluorescent signals and prevent photobleaching. It contains the DNA-binding dye DAPI, which stains nuclei.
Automatically generated - may contain errors
Lab products found in correlation
Vector trueview autofluorescence quenching kit, by Vector Laboratories (1 mentions)
Fv1000 confocal microscope, by Olympus (1 mentions)
Rabbit anti c3, by Abcam (1 mentions)
Goat anti gfap, by Abcam (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Rabbit anti iba1, by Fujifilm (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Rat anti cd68, by Bio-Rad (1 mentions)
Stellaris 5 confocal laser scanning microscope, by Leica (1 mentions)
3 protocols using vibrance antifade mounting medium with dapi
1
Immunofluorescence Imaging of Brain Tissue
2
Immunofluorescence of Retinal Cryosections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cryosections (12 μm) were incubated at room temperature in a blocking solution (5 % normal donkey serum, 0.3 % Triton X-100 in PBS) for 1 h. Subsequently, retinal sections were incubated with primary antibodies at 4 °C overnight. The following primary antibodies were used: rabbit anti-C3 (Abcam, Cambridge, UK), rabbit anti-IBA1 (Wako, Osaka, Japan), rat anti-CD68 (Bio-Rad, Hercules, CA, USA), goat anti-GFAP (Abcam), and mouse anti-FDP-lysine (ACR-modified protein; a gift from Dr. Uchida, University of Tokyo, Japan), mouse anti-GS (glutamine synthase; Abcam) antibody. After washing, sections were incubated with secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 594 (Invitrogen) for 1 h at room temperature. Sections were mounted using Vibrance Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA) and analyzed using BZ-X810.
3
Immunofluorescence Imaging of Tissue Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescence was performed on tissues which were age‐matched to our flow cytometry samples. Formaline fixed paraffin embedded (FFPE) tissue or fresh frozen samples which were embedded in optimal cutting temperature, were used. For FFPE material, 6 micron sections were deparaffinized in xylene and rehydrated in a graded ethanol series. Antigen retrieval was performed for 15 min at 94–96 °C on a hot plate in 10mM sodium citrate at pH 6. Antigen retrieval was performed in a similar fashion for frozen sections (6 micron) for 5 min. Primary antibodies were diluted in PBS and sections were incubated for 1.5 h. Sections were washed in PBS and incubated for 1 h with secondary antibodies. Sections were washed and mounted using Vectashield Vibrance Antifade Mounting Medium with DAPI (Vectorlabs, H1800‐10). Images were taken on a Leica Stellaris 5 confocal laser scanning microscope with the 20x objective. Antibodies used are listed in Table 1 .
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!