Cells were fixed on coverslips with 4% formaldehyde for 15 min at room temperature. Upon permeabilization, the cells were blocked with 0.1% Triton X-100 in 4% normal goat serum in PBS for 1 h. Immunofluorescence staining was carried out with primary antibody against GRP75 (1:1000 # ab53098; Abcam) overnight at 4 °C. Secondary fluorescence antibody Goat anti-Rabbit IgG Alexa Fluor 488 (1:400; Invitrogen), was applied for 1 h at room temperature. The cells were mounted with DAPI-Fluoromount-G® (Southern Biotech), and the images were taken as Z-stacks using a confocal microscope. At least fifteen cells from two coverslips per individual were analysed using ImageJ (NIH software; Rasband, W.S., ImageJ, U. S. National Institute of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/ , 1997–2018) as described previously48 (link).
Ab53098
Manufactured by Abcam
Sourced in United States
Ab53098 is a laboratory equipment product. It is a device designed for use in scientific research applications. The core function of this product is to facilitate specific experimental procedures or analytical tasks, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 goat anti rabbit igg h c, by Thermo Fisher Scientific (2 mentions)
Bx53 microscope, by Olympus (2 mentions)
Antifade mounting medium, by Beyotime (2 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Dapi fluoromount g, by Southern Biotech (1 mentions)
Dako lsab kit, by Agilent Technologies (1 mentions)
Silane coated slides, by Merck Group (1 mentions)
Ab2799, by Abcam (1 mentions)
Ab21685, by Merck Group (1 mentions)
Ab104223, by Abcam (1 mentions)
Ab144p, by Merck Group (1 mentions)
Fluoview fv1000, by Olympus (1 mentions)
Albumin bovine 5, by Solarbio (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
4 6 diamidino 2 phenylindole dapi c1006, by Beyotime (1 mentions)
6 protocols using ab53098
1
Immunofluorescence Quantification of GRP75
2
Immunofluorescence Staining of Adipose Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For Immunofluorescence, 4 μm sections were stained against GRP75 (Antibodies Inc, 75–127 or Abcam ab53098), PLIN1 (Abcam ab61682), and COX4 (Novex 459600) using appropriate secondary antibodies conjugated to Alexa Fluor 488 (COX4), Alexa Fluor 568 (PLIN1), or Alexa Fluor 647 (GRP75), and DAPI as nuclear stain, as described previously [14 ].
Whole-mount preparations were generated from small pieces (<5 mm) of formalin-fixed pADR adipose tissue. All staining procedures took place at 4 °C in a reaction tube on a rotating rack. Briefly, tissues were blocked in a solution of PBS, 1 % Triton X-100, 2 % BSA, 5 % normal donkey serum, 0.1 % sodium azide for 2 h, afterwards the solution was replaced with blocking buffer containing antibody against GRP75 for 48 h. Subsequently, tissues were washed 3 times with PBS, 1 % Triton, 30 min per wash, and incubated with a fluorescent conjugated secondary antibody, 1 μg/mL BODIPY493/503 (BD493), and DAPI in blocking buffer for 72–96 h. Following a final wash step, tissues were equilibrated in 70 % glycerol and mounted between two #1.5 glass coverslips separated by a 1 mm deep FastWell™ incubation chamber (Grace Biolabs Inc., Bend, OR) for imaging purposes.
Whole-mount preparations were generated from small pieces (<5 mm) of formalin-fixed pADR adipose tissue. All staining procedures took place at 4 °C in a reaction tube on a rotating rack. Briefly, tissues were blocked in a solution of PBS, 1 % Triton X-100, 2 % BSA, 5 % normal donkey serum, 0.1 % sodium azide for 2 h, afterwards the solution was replaced with blocking buffer containing antibody against GRP75 for 48 h. Subsequently, tissues were washed 3 times with PBS, 1 % Triton, 30 min per wash, and incubated with a fluorescent conjugated secondary antibody, 1 μg/mL BODIPY493/503 (BD493), and DAPI in blocking buffer for 72–96 h. Following a final wash step, tissues were equilibrated in 70 % glycerol and mounted between two #1.5 glass coverslips separated by a 1 mm deep FastWell™ incubation chamber (Grace Biolabs Inc., Bend, OR) for imaging purposes.
3
Immunohistochemical Staining of Mortalin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Dako LSAB kit (Dako, Glostrup, Denmark) was used for IHC. Serial 4 μm-thick tissue sections were prepared on silane-coated slides (Sigma, St. Louis, MO, USA), and deparaffinized, rehydrated and incubated with 3 % H2O2 in methanol for 10 min at room temperature to eliminate endogenous peroxidase activity. The antigen was retrieved at 95 °C for 20 min by placing the slides in 0.01 M sodium citrate buffer (pH 6.0). Slides were then incubated with the primary antibody of rabbit anti-Mortalin antibody (ab53098) (Abcam, Cambridge, MA, USA), at 4 °C overnight. After incubation at room temperature for 30 min with biotinylated secondary antibody, slides were incubated with streptavidin–peroxidase complex at room temperature for 30 min. Slides were immunostained with 3,3’-diaminobenzidine chromogen and then counterstained with Mayer’s hematoxylin. We used tonsil sections as the positive control and Rabbit IgG as an isotope control. In addition, tissue sections were processed omitting the primary antibody as the negative control.
4
Immunocytochemistry of iMN Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
iMNs plated on coverslips were fixed using 4% paraformaldehyde (PFA) for 15 min and blocked for 1 h with 3% bovine serum albumin (BSA) and 0.1% TritonX-100 in phosphate buffered saline PBS. iMNs were incubated with the following primary antibody: mouse anti-GRP75 (Abcam, ab2799, 1:200), rabbit anti-GRP75 (1:200, Abcam, ab53098), rabbit anti-BiP (Abcam, ab21685, 1:500), goat anti-ChAT (Millipore, AB144P, 1:500), chicken anti-MAP2 (Sigma Aldrich, AB15452, 1:500), rat anti Isl1-2 (DSHB 39.4D5), mouse anti-HB9 (1:500, DSHB 81.5C10), mouse anti-TDP43 (ABCAM ab104223, 1:500), in blocking buffer overnight at 4 °C. After three washes with PBS, cells were incubated in blocking buffer with appropriate Alexa Fluor secondary antibodies and DAPI for 1 h at RT, followed by three PBS washes and coverslips were mounted on glass slides. Confocal images were acquired with FluoViewTM FV1000 (Olympus) fitted with a 20 × or 40 × air objective and 60 × immersion oil objective.
5
Immunofluorescence Assay of Mortalin in Breast Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Breast cancer MDA-MB231 cells were grown on coverslips to 70 % confluence. The cells were fixed with 4 % paraformaldehyde for 10 min, and after 24 h cells were permeabilized with 0.5 % TritonX-100 for 10 min. Blocking was performed with 3 % Albumin Bovine V (A8020, Solarbio, Beijing, China) for 1 h at room temperature. After washing with PBS, cells were incubated with rabbit anti-Mortalin antibody (ab53098) (Abcam, Cambridge, MA, USA), at 4 °C overnight, followed by incubation with Alexa Fluor 488 Goat Anti-Rabbit IgG (H + C) (A11008, 1:1000, Invitrogen, USA) for 1 h at room temperature. After washing with PBS, the cells were counterstained with DAPI (C1006, Beyotime, Shanghai, China), and the coverslips were mounted with an Anti fade Mounting Medium (P0126, Beyotime, Shanghai, China). IF signals were visualized and recorded with a BX53 Olympus microscope.
6
Mortalin Immunofluorescence in A549 NSCLC Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The A549 NSCLC cell line was grown on coverslips to 70%-80% confluence, and then all of the cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.5% Triton X-100 for 10 min at room temperature. Subsequently, after blocking with 3% bovine albumin fraction V (A8020; Solarbio, Beijing, China) for 1 h and washing with phosphate-buffered saline (PBS), the cells were incubated with rabbit anti-Mortalin antibody (ab53098; Abcam, Cambridge, MA, USA), at 4°C overnight and then incubated with Alexa Fluor ® 488 Goat Anti-Rabbit IgG (H+C) (A11008, 1:1000; Invitrogen, CA, USA) for 1 h. Next, the cells were washed with PBS and counterstained with 4,6-diamidino-2-phenylindole (DAPI; C1006, Beyotime, Shanghai, China). The coverslips were mounted with Antifade Mounting Medium (P0126, Beyotime, Shanghai, China). Finally, the immunofluorescence (IF) signals were visualized and recorded using a BX53 Olympus microscope.
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!