Immunocytochemistry was performed on day 10–11 after plating the cells on the PET 5 textile samples. The samples were fixed with 4% paraformaldehyde, blocked with 10% normal donkey serum (Biowest, Nuaille, France) solution, and stained with goat anti-cardiac troponin T (1:1,000, Abcam) and mouse anti-MyBPC3 (1:400, Santa Cruz Biotechnology, Dallas, Texas, USA) at 4 °C overnight. Donkey anti-goat Alexa Fluor 568 and donkey anti-mouse Alexa Fluor 488 (1:800, Thermo Fisher Scientific, Waltham, Massachusetts, USA) were used as secondary antibodies. The cell nuclei were stained using Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, California, USA). Fluorescence was visualized with a Nikon A1R+ Laser Scanning Confocal Microscope (Nikon, Tokyo, Japan) using a Nikon Apo 60× 1.40NA oil objective and with Zeiss Axio Imager.M2 with ApoTome.2 and AxioCamHRm3 camera using a Zeiss EC Plan-Neofluar 40× 1.30NA oil objective.
Normal donkey serum (nds)
Manufactured by Biowest
Sourced in France
Normal donkey serum is a biological product derived from the blood of healthy donkeys. It serves as a source of various proteins, growth factors, and other biomolecules found in the serum of donkeys.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100 pbs, by Merck Group (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Hoechst 33258, by Merck Group (2 mentions)
Axio imager m2, by Zeiss (1 mentions)
Anti mouse alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (1 mentions)
A1r laser scanning confocal microscope, by Nikon (1 mentions)
Ec plan neofluar 40, by Zeiss (1 mentions)
Donkey anti goat alexa fluor568, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 donkey anti rat igg, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Biowest (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
4 protocols using normal donkey serum (nds)
1
Immunocytochemistry of Cardiac Cells
2
Immunohistochemistry of Cryosectioned Brains
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cryosectioned brains were air-dried for 1 h at RT, followed by rehydration in PBS (Thermo Fisher Scientific) for 10 min. After antigen retrieval the sections were permeabilized in 0.25% Triton X-100/PBS (Sigma-Aldrich) for 10 min followed by washing in PBS. The brain tissue was blocked in the blocking buffer [10% Normal donkey serum (Biowest) 2% BSA (Sigma-Aldrich)] at RT for 1 h. Sections were incubated with the diluted primary antibody (Supplementary Table 3 ) ON at 4C followed by washing in PBS 3 times for 5 min each. The sections were then incubated in diluted secondary antibody for 2 h at RT and washed in PBS for 3 times for 5 min each. The sections were counterstained with 10 g/mL Hoechst 33258 (Sigma-Aldrich) for 10 min followed by washing in PBS for 5 min twice. Secondary antibodies were conjugated with either Alexa fluorophores, 488, 594 or 647 and diluted at 1:500 according to the manufacturers recommendations (Supplementary Table 4 ). Negative controls were performed by using IgG controls (Supplementary Table 4 ) and by omitting the primary antibody.
3
Visualizing Retinal Vasculature and Apoptosis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Retinas were dissected and stained as described with minor modifications (Zarkada et al., 2015 (link)). Briefly, for iB4 stainings, the retinas were equilibrated 30 min in PBlec (0.1 mM CaCl2, 0.1 mM MgCl2, 0.1 mM MnCl2, and 1% Triton X-100 in PBS, pH 6.8) and incubated with biotinylated Griffonia simplicifolia lectin I iB4 (1:25; Vector; CAT#B-1205) diluted in PBlec, overnight at 4°C. iB4 was detected by incubation with streptavidin-conjugated Alexa Fluor 488 (1:200; Invitrogen; CAT#S-32354) overnight. For iB4/ERG/cCasp3 combination stainings shown in Fig. 4 , following iB4 staining and post-fixation in 1% PFA in PBS for 5 min at room temperature, the retinas were washed with PBS for 30 min, blocked with donkey immunomix (DIM; 5% normal donkey serum [Biowest], 2% BSA [Biowest], and 0.3% Triton X-100 [Sigma-Aldrich] in PBS) for 1 h at room temperature and incubated with rabbit anti-cCasp3 (1:300; Cell Signaling Technology; CAT#9664) diluted in DIM overnight at 4°C, which was detected with Alexa Fluor 594–donkey anti-rat IgG (1:500; Invitrogen; CAT#A-21209). The retinas were then washed all day in PBS and incubated with rabbit-anti-ERG-Alexa 647 conjugated antibody (1:100; Abcam; CAT#ab196149) for 2 d before post-fixation and mounting.
4
Immunofluorescence staining of brain tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cryosectioned brains were air-dried for 1hr at RT, followed by rehydration in PBS (Thermo Fisher Scientific) for 10 min. After antigen retrieval the sections were permeabilized in 0.25% Triton X-100/PBS (Sigma-Aldrich) for 10 minutes followed by washing in PBS. The brain tissue was blocked in the blocking buffer (10% Normal donkey serum (Biowest) 2% BSA (Sigma-Aldrich)) at RT for 1 h. Sections were incubated with the diluted primary antibody (see below) ON at 4°C followed by washing in PBS 3 times for 5 mins each. The sections were then incubated in diluted secondary antibody for 2h at RT and washed in PBS for 3 times for 5 mins each. The sections were counterstained with 10 µg/ml Hoechst 33258 (Sigma-Aldrich) for 10 min followed by washing in PBS for 5 mins twice. Secondary antibodies were conjugated with either Alexa fluorophores, 488, 594 or 647.
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!