Paraffin sectioned from adult SFD and age-matched control globes were de-paraffinized and antigen retrieval performed using 10mM sodium citrate buffer with 0.05% Tween-20. Tissues were blocked with 10% horse serum and incubated overnight with primary antibodies at 4°C. Antibodies included ApoE (1:1000, AB947), Bestrophin (1:250, MAB5466, Millipore), Collagen VI (1:250, ab6588, Abcam, Cambridge, MA), Clusterin (1:200, AB825, Millipore), CRALBP (1:1000, gift from Dr. Jack Saari, UW Vision Core, Seattle, WA), TIMP2 (1:500, H00007077-M03J, Novus Biologicals, Centennial, CO), TIMP3 (1:5000, GTX25939, GeneTex, Irvine, CA), and Vitronectin (1:1000, AB19014, Millipore). Alexa Fluor secondary antibody (Molecular Probes, Grand Island, NY) were used at 1:500 dilution. Sections were counterstained with DAPI (Sigma-Aldrich) and mounted with Fluoromount-G (SouthernBiotech, Birmingham, AL, USA). Images were taken using a 63x objective lens with the Leica DM6000 CS confocal microscope (Leica Microsystems). For iPSC- RPE cells, filter inserts were fixed in 4% PFA, entered a sucrose gradient, excised from the plastic holder and embedded in Tissue-TEK OCT compound (Sakura Finetek USA, Inc, Torrance, VA, USA). Blocks were cut on the Leica CM1850 cryostat to obtain 20 μm thick sections. Staining was performed as described above.
Dm6000 cs confocal microscope
Manufactured by Leica
Sourced in Germany
The Leica DM6000 CS is a confocal microscope designed for high-resolution imaging. It features a motorized stage, a high-performance optical system, and advanced software for image acquisition and analysis. The DM6000 CS is capable of capturing detailed, three-dimensional images of samples with minimal out-of-focus light.
Automatically generated - may contain errors
Lab products found in correlation
Duolink in situ detection reagents orange, by Merck Group (2 mentions)
Tissue tek oct compound, by Sakura Finetek (1 mentions)
Cm1850 cryostat, by Leica (1 mentions)
Alexa fluor secondary antibody, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Mab5466, by Merck Group (1 mentions)
Vitronectin, by Merck Group (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
Optimal cutting temperature compound, by Sakura Finetek (1 mentions)
Ab28481, by Abcam (1 mentions)
Acc 049, by Alomone (1 mentions)
Anti gfp, by Thermo Fisher Scientific (1 mentions)
Tcs sp8 confocal laser scanning microscope, by Leica (1 mentions)
7 protocols using dm6000 cs confocal microscope
1
Immunohistochemical Profiling of Retinal Tissues
2
Immunofluorescence Analysis of Hepatic SREBP1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescence staining of the liver tissues was performed under cryoprotection as previously described with minor modifications (19 (link)). The mouse livers were excised, fixed in 4% paraformaldehyde and 30% sucrose solution, and processed for embedding with optimal cutting temperature compound (Sakura Finetek, Tokyo, Japan). Cryosectioning was performed at −20°C, and the frozen liver tissues were sectioned into 5-μm-thick slices followed by mounting on glass slides. For immunofluorescence staining, the sections were air-dried on a bench for 10 min, and were blocked in phosphate-buffered saline (PBS) containing 0.1% BSA and incubated overnight at 4°C with an anti-SREBP1 antibody (ab28481; Abcam, Cambridge, MA, USA) at a dilution of 1/200 followed by incubation with Alexa-conjugated goat anti-rabbit secondary antibody (#CA11008s; Invitrogen, Carlsbad, CA, USA) for 1 h. Following 3 rinses with PBS, the sections were mounted with PBS with 5 μg/ml of 4′,6-damidino-2-phenylindole dihydrochloride (DAPI; Sigma-Aldrich) and the specimens were imaged under a Leica DM6000 CS confocal microscope (Leica Microsystems, Wetzlar, Germany).
3
Quantification of Foxp3 and β-Catenin Interaction
4
Immunofluorescence Quantification of TRPM8
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were grown on coverslips, fixed with 4% PFA and permeabilized in 5% FBS/0.1% Triton X-100. Afterward, cells were blocked and incubated with primary antibodies overnight at 4°C (see Supplementary Table S4 ). After washing, cells were incubated with Alexa Fluor conjugated secondary antibodies and counterstained with DAPI. For the semiquantitative analysis of TRPM8 expression, we adopted a nonpermeabilizing protocol to detect only its localization at the plasma membrane. Cells grown on coverslips were washed with ice-cold 2% BSA in PBS and incubated with anti-TRPM8 (Alomone Labs, ACC-049) and anti-E-Cadherin antibodies for 1 h at 4 °C. After washing, cells were incubated with Alexa Fluor conjugated secondary antibodies, fixed with 4% PFA and counterstained with DAPI. For the quantification of plasma membrane associated TRPM8 channels, TRPM8 dots from more than 6000 cells were counted by two independent researchers. All the images were acquired using a Leica DM6000 CS confocal microscope. Immunofluorescence studies were performed in at least three independent biological replicates; representative data are shown.
5
DNA Patterning on Gold Surfaces
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To develop the pattern on gold surface, FAM- or TAMRA-modified reporter (RS) DNA was incubated on the surface. For the pattern with one color, 2 μM 3′ FAM-modified RS DNA was incubated with surface for 1 h. For the pattern with two different colors, a 2 μM mixture of 3′ FAM-modified RS DNA and 3′ TAMRA-modified RS DNA (RS-1) was incubated on the surface for 1 h to hybridize with linking-DNA sequences. After pattern development, a Leica DM6000 CS confocal microscope (TCS SP5 II) was used to take all fluorescent images.
6
Quantification of Foxp3 and β-Catenin Interaction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PLA was performed with Duolink In Situ Detection Reagents Orange (Sigma) according to the manufacturer’s recommendation with minor modifications. Treg cells were cultured for four days and harvested, and cells were fixed with 2% paraformaldehyde for 10 min at RT. Fixed cells were incubated in Foxp3 Fix/Perm buffer set for 30 min at 4°C, followed by staining with mouse anti-β-catenin (14/Beta-Catenin) and rabbit anti-Foxo1 (C29H4) for 1 h at RT in Foxp3 staining buffer. Cells were washed and stained in Foxp3 staining buffer with the secondary mouse PLUS and rabbit MINUS antibodies for 30 min at RT. Cells were washed in TBS (0.01 M Tris, 0.15 M NaCl) with 0.5% BSA and the ligation reaction was performed at 37°C for 30 min, followed by the amplification reaction at 37°C for 100 min. Cells were washed in TBS (0.2 M Tris, 0.1 M NaCl) with 0.5% BSA and stained with anti-Foxp3 (PCH101) or anti-IFN-γ (B27) antibody for 30 min at 4°C. Cells were analyzed with a 60x or 100x objective on a Leica DM6000 CS confocal microscope.
7
Immunohistochemistry in Adult and Pupal Brains
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the immunohistochemistry in adult and pupal brains, the following protocol was used: Brains were collected in cold PBS and subsequently fixed for 10 min in 4% formaldehyde (in PBS 0.3% Triton X‐100). Upon three washes in PBS 0.3% Triton X‐100, the brains were blocked in 10% BSA (in PBS 0.3% Triton X‐100) for 1 h rocking at RT. After this step, the brains were incubated overnight at 4°C with the primary antibody appropriately diluted in blocking solution. The second day, the samples were washed 3 times in PBS 0.3% Triton X‐100 and then incubated with the appropriate secondary antibody diluted in blocking solution for 1h at RT. Upon three washes in PBS 0.3% Triton X‐100, the brains were mounted and kept at 4°C for imaging. Primary antibody used were as follows: anti‐FasII 1/50 (ID4 Hybridoma Bank) and anti‐GFP 1/500 (Invitrogen A11122). Secondary antibody used were Alexa Fluor 555 Donkey anti‐Mouse and Alexa Fluor 488 Goat Anti‐Rabbit at a dilution of 1/500. Images were acquired on a Leica TCS SP8 laser scanning confocal microscope or on a Leica DM6000CS confocal microscope and processed using Fiji™ software.
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!