www.nature.com/scientificreports OPEN Characterization of microvessels in the human forehead dermis using intravascular dual perfusion and immunofluorescence staining Shu Rui, Zilong Cao,Yunzhang Wang &
As with standardIHC, primary or direct IF uses a single Ab that is chemically linked to a fluorophore. Direct conjugation to fluorophore reduces the number of steps in the staining procedure making the process faster and reducing background signal by avoiding issues with Ab cross-reactivity or no...
The final staining sequence is shown in Table 4. The complete staining protocol is presented in Table A.2. The fluorophore manufacturer and some authors recommend two applications of fluorophores when conducting automated immunofluorescence staining [17], [40]. This strategy is referred to as ‘doub...
Secondary antibodies included Alexa Fluor 488 (1:200 dilution; Abcam, USA) for β-tubulin and Alexa Fluor 594 (1:200 dilution; Abcam, USA) for vimentin. Incubation time was 1 h, at 37 °C, then cells were washed in 1X PBS (3 times) and finally slides were mounted with ProLong™ ...
Molecular weights of each protein were determined using ImageJ software, with GAPDH staining serving as a control for the loading of protein within each well of the western blot. range of the IF assays to detect MCT1 and MCT4 in cancer cells. A range of antibody concentrations were in- ...
The whole section cohort of MPM samples were stained following the same conditions as the optimized mIF protocol. Similarly, we included a positive control (tonsil) and autofluorescence tissue (negative controls) during the staining of the cases. The slides were scanned using the Vectra/Polaris 3.0...
For optimal performance, samples were imaged within 2 weeks of clearing; however, staining was still observed up to 6 months after clearing. Diffuse, non-specific fluorescence was observed using the SeeDB protocol in the absence of the primary antibodies (Additional file 1: Figure S1, bottom ...
Molecular weights of each protein were determined using ImageJ software, with GAPDH staining serving as a control for the loading of protein within each well of the western blot. Full size image Optimisation of staining intensity Flow cytometry was employed together with the two control cell lines...
23,25 The choice of fluorophore for each antibody is also critical and has to be optimized for each specific tissue type, as the spectral unmixing protocol is influenced by the autofluorescence present in the tissue.25 Importantly, the validity and reproducibility of the staining protocol can be ...
Furthermore, it details a more cost-effective protocol for antigen retrieval and immuno-staining when generating high-resolution 3-D reconstructions. Finally, it is detailed how to perform image acquisition by multiple imaging modalities for 3-D reconstruction, and how it is possible to quantify ...