HeLa cells growing on six-well chamber slides were washed once with PBS, extracted with CSK buffer [10 mM Pipes (pH 7.0), 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, and 0.5% Triton X-100] for 2 min, washed again with cold PBS, and fixed in 4% (w/v) paraformaldehyde for 10 min. Cells were permeabilized with 0.2% Triton X-100 in PBS, blocked with 0.8% bovine serum albumin (BSA), and incubated with appropriate primary antibodies coupled to Alexa Fluor 488 (Jackson ImmunoResearch; rabbit, 111-545-003, 1:100), Alexa Fluor 594 (Jackson ImmunoResearch; mouse, 115-585-003, 1:100), Alexa Fluor 488 (Jackson ImmunoResearch; mouse, 115-545-003, 1:100), or Alexa Fluor 555 (Life Technologies; rat, A21434, 1:100, for BrdU staining). Cells were then washed for four times, and a final concentration of 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (0.1 μg/ml; Sigma) was included in the final wash to stain nuclei. Confocal laser scanning microscopy images were obtained using a Zeiss LSM 510 microscope equipped with argon laser (365 nm, 20-Hz pulse). The images were captured and analyzed using Adobe Photoshop CS4.
Alexa fluor 488
Manufactured by Jackson ImmunoResearch
Sourced in United States, United Kingdom, Panama, China, Germany, Cameroon
Alexa Fluor 488 is a fluorescent dye manufactured by Jackson ImmunoResearch. It is a small-molecule fluorophore designed for use in various biological and immunological applications. The dye exhibits bright green fluorescence when excited by a 488 nm light source.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 594, by Jackson ImmunoResearch (68 mentions)
Alexa fluor 647, by Jackson ImmunoResearch (51 mentions)
Cyanine3 (cy3), by Jackson ImmunoResearch (40 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (22 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (21 mentions)
Hoechst 33342, by Thermo Fisher Scientific (15 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (11 mentions)
Lsm 710, by Zeiss (10 mentions)
Vectashield, by Vector Laboratories (9 mentions)
Alexa fluor 568, by Jackson ImmunoResearch (9 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (8 mentions)
Lsm 510, by Zeiss (8 mentions)
Alexa fluor 555, by Jackson ImmunoResearch (7 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (7 mentions)
Bovine serum albumin (bsa), by Merck Group (7 mentions)
Rhodamine red x, by Jackson ImmunoResearch (6 mentions)
Lsm 700, by Zeiss (6 mentions)
Mowiol, by Merck Group (6 mentions)
Hoechst, by Thermo Fisher Scientific (6 mentions)
Anti gfp, by Abcam (5 mentions)
424 protocols using alexa fluor 488
1
Immunofluorescence Staining of HeLa Cells
2
Immunofluorescent Staining of Cells
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The cells were plated on coverslips and allowed to grow before being fixed with 4% PFA at room temperature for 30 min. Subsequently, the coverslips were blocked with PBS containing 5% donkey serum at room temperature for 30 min. The cells were then incubated with primary antibodies overnight at 4 °C. Following the removal of the primary antibodies, the sections were washed in PBS and stained with secondary antibodies for 45 min at room temperature. The nuclei were counterstained with DAPI before mounting the coverslips on glass slides.
The following primary antibodies were used for immunostaining: CD31 (Abcam, ab28364, 1:100 for coverslips or R&D Systems, FAB3628G, 1:100 for bone sections), Endomucin (Santa Cruz, sc-65495, 1:200), alpha smooth muscle actin (Abcam, ab124964, 1:400), CD45 (BD Pharmingen, 557659, 1:200), and RUNX2 (Cell Signaling Technology, 12556 S, 1:400).
The following secondary antibodies were used in immunostaining (all obtained from Jackson ImmunoResearch): donkey anti-rabbit Alexa Fluor 488 (711-545-152, 1:400) and Alexa Fluor 647 (711-605-152, 1:400); donkey anti-rat Alexa Fluor 488 (712-545-150, 1:400), Alexa Fluor 594 (712-585-150, 1:400), and Alexa Fluor 647 (712-605-150, 1:400); and donkey anti-goat Alexa Fluor 488 (705-545-147, 1:400).
The following primary antibodies were used for immunostaining: CD31 (Abcam, ab28364, 1:100 for coverslips or R&D Systems, FAB3628G, 1:100 for bone sections), Endomucin (Santa Cruz, sc-65495, 1:200), alpha smooth muscle actin (Abcam, ab124964, 1:400), CD45 (BD Pharmingen, 557659, 1:200), and RUNX2 (Cell Signaling Technology, 12556 S, 1:400).
The following secondary antibodies were used in immunostaining (all obtained from Jackson ImmunoResearch): donkey anti-rabbit Alexa Fluor 488 (711-545-152, 1:400) and Alexa Fluor 647 (711-605-152, 1:400); donkey anti-rat Alexa Fluor 488 (712-545-150, 1:400), Alexa Fluor 594 (712-585-150, 1:400), and Alexa Fluor 647 (712-605-150, 1:400); and donkey anti-goat Alexa Fluor 488 (705-545-147, 1:400).
3
Immunohistochemical Analysis of Cholinergic Neurons
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Adult mice were anesthetized with an overdose of pentobarbital and then transcardially perfused with 4% paraformaldehyde (PFA). Mouse brain was dissected and fixed in 4% PFA for 4 h. After cryoprotection in 30% sucrose, brain sections (35 μm) were cut on a cryostat microtome (Leica CM1950, Leica Biosystems, Wetzlar, Germany). After rinsing with PBS and 0.3% Triton-X in 0.1 M PBS (PBST), the brain sections were blocked with 2% (w/v) bovine serum (BSA) in PBST for 1 h. Then, the brain sections were incubated with primary antibodies at 4°C for 48 h and secondary antibodies at room temperature for 2 h. Images were collected using a Zeiss LSM510 Meta or Nikon A1 confocal microscope and analyzed using FIJI. The antibodies used were as follows: anti-choline acetyltransferase (1:200, goat, AB144P, MERCK, Kenilworth, NJ, United States), anti-NK1R (S8305, rabbit, 1:5,000, Sigma-Aldrich, St. Louis, MO, United States), goat anti-rabbit for NK1R (Cy3 and Alexa Fluor 488, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, United States), donkey anti-goat for choline acetyltransferase (Cy3 and Alexa Fluor 488, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, United States), and Cy3-streptavidin (1:500).
4
Immunohistochemical Analysis of Mouse Brain
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Mice were perfused with 0.01 M PBS, and 4% freshly made paraformaldehyde under deep anesthesia53 (link). Coronal brain sections were cut at 10 μm thickness using a cryostat microtome (CM1950, Leica). After post-fixation in 4% paraformaldehyde for 15 min, sections were washed with PBS for 5 min, permeabilized in TBS containing 0.3% Triton X-100 for 20 min, and blocked in TBST (0.1% Tween) containing 5% BSA, 0.3% Triton X-100 for 1 h at room temperature. Sections were incubated with the primary antibody overnight at 4 °C in TBST with 1% BSA, 0.3%Triton X-100. Primary antibodies (anti-Iba1: 1:250, ab178847; anti-GFAP: 1:500, ab7260; anti- Parvalbumin: 1:500, ab11427) were visualized with an appropriate secondary antibody (goat Anti-Rabbit IgG H&L (Alexa Fluor® 488), 1:1000~1500, ab150077) conjugated with Alexa-Fluor 488 (Jackson Immunoresearch) after washing 3 times with TBST. Slides were covered using a coverslip under the Fluoroshield Mounting Medium spiked with DAPI (Abcam; ab104139). Images were acquired using a fluorescence microscope (M2, Zeiss), a confocal microscope (LSM 710; Zeiss), and quantified using Image J.
5
Immunofluorescent Staining of UFM1 and FAT10
6
Quantifying Pancreatic Islet Composition
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The splenic lobe of the pancreases from all STZ-diabetic rats, 5 Non-D Chow and 5 Non-D Keto rats were dissected, paraffin embedded and sectioned at 5 µm on a microtome (Ergostar Microm HM200, Microm, Walldorf, Germany). The sections were mounted on glass slides and stained for insulin (1:100, #4590S rabbit polyclonal anti-insulin antibody, Cell Signalling Technology, Danvers, MA, USA) and glucagon (1:2000, #G2654 mouse monoclonal anti-glucagon antibody, Sigma-Aldrich, Macquarie Park, NSW, Australia). The corresponding fluorophore-conjugated secondary antibodies were donkey anti-rabbit AlexaFluor488 (1:500, 711-546-152, Jackson Immunoresearch, West Grove, PA, USA) and donkey anti-mouse Cy5 (1:500, 715-175-151, Jackson Immunoresearch, West Grove, PA, USA), respectively. Pancreatic islets (8–12 per animal) were visualised with Zeiss Axio Imager Z2 fluorescence microscope (Zeiss, Germany) under 10× objective. The ratio of insulin-positive to glucagon-positive staining within each islet was determined in FIJI [23 (link)] by measuring the thresholded area of each channel by an operator blinded to experimental conditions.
7
Pupal Eye Immunostaining Protocol
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Pupal eyes were dissected and fixed as previously described (DeAngelis and Johnson 2019 ). For 1° antibody staining, rat anti-E-cad (1:20, DSHB, # 528120) was used to visualize cell boundaries. Secondary antibodies conjugated to Alexa Fluor® 488 (Jackson ImmunoResearch) were used at 1:200. Dissected pupal eyes were imaged with a Leica DM5500 B fluorescence microscope and corresponding software.
8
Immunofluorescence Quantification of Opsin and Recoverin
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Cells cultured in glass coverslip were fixed with 4% paraformaldehyde in PBS pH 7.4 for 10 min. at room temperature. After fixation, the cultures were washed with PBS and incubated 1 hr with blocking solution 5% BSA serum (Sigma‐Aldrich) 0.05% Tween in PBS, and then incubated overnight with monoclonal anti‐opsin (1:1000; Sigma‐Aldrich) and anti‐recoverin (1:1000; Abcam, Cambridge, UK) antibodies. Alexa Fluor® 488 (Jackson Laboratory, West Baltimore Pike, West Grove, PA, USA)‐conjugated goat polyclonal antibody (1:1000) was used as secondary for opsin detection. Cy3‐conjugated goat polyclonal anti‐rabbit (Jackson Laboratories; 1:400) was used as secondary for recoverin detection. Nuclei were counterstained by DAPI. Quantification of fluorescence intensity was determined by LEICA software (Milan, Italy). The method used by Alessio et al. 24 was applied to calculate the percentage of positive cells in each microscope field. This was calculated by the number of green or red (opsin or recoverin) positive cells of 400 cells in six different microscope fields according to the previous method 24 .
9
Dual Immunostaining of Adherent Cells
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Cells were planted on 24-well plates chamber slides and grew overnight to adhere. Dual immunostaining was performed sequentially. First, the cells were fixed with cooled 4% paraformaldehyde for 30 min and permeabilized with 0.25% Triton X-100 for 1 h. After being blocked with 5% BSA for 1 h, the cells were incubated in 5% BSA at 4 °C overnight with the primary antibody. Next, the cells were washed with PBS twice and incubated in 5% BSA for 1 h at room temperature with secondary antibodies Alexa Fluor 488 (Lot:12194) and Cy3(Lot:125099) from Jackson ImmunoResearch (USA). Nuclei were stained with Hoechst (Beyotime, 33342, Shanghai, China). Photographs were taken using a confocal microscope (Carl Zeiss Microscopy GmbH, Germany).
10
Immunofluorescence and IHC Staining Protocol
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For immunofluorescence staining, paraffin slides were first deparaffinized with xylene, then hydrated in a series of graded ethanol, and finally with water. Antigen retrieval was then performed in a Tris-HCl buffer (pH = 9.0) using a pressure cooker. The tissues were blocked with a blocking buffer (1X PBS containing 3% donkey serum and 0.3% Triton X-100) for one hour. Primary antibodies, listed in Supplementary Table 1 , were diluted in the blocking buffer at a ratio of 1:200. The diluted primary antibodies were added to the tissues, which were then stored in a 4 °C refrigerator overnight. The next day, tissues were washed with 1X PBS for 30 min. Secondary antibodies conjugated to Alexa Fluor 488, Cy3, or Cy5 (Jackson ImmunoResearch Laboratories) were diluted in 1X PBS and added to the slides. ImageJ (National Institutes of Health) (v1.53c) was used for the quantitative analysis of the immunofluorescence-stained images. For IHC staining, following primary incubation, tissue sections were incubated with secondary antibodies conjugated to HRP and then reacted with DAB substrates. Secondary antibodies are listed in Supplementary Table 2 .
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