Brain sections (5 μm) were fixed in 10% buffered formalin for 24 h and then embedded in paraffin following standard protocols. For immunohistochemical analysis, paraffin was removed and the tissues were rehydrated and boiled for 2 min in citrate buffer. After, samples were incubated 10 min with ammonium chloride. Subsequently, tissue sections were incubated 10 min with PBS/Triton X-100 0.1% and further incubated for 20 min with PBS/BSA 2%. Sections were then incubated overnight at 4oC with mouse monoclonal antibody raised against human α-tubulin at a 1:50 dilution, or rabbit polyclonal antibody raised against proteins obtained from C. glabrata at a 1:500 dilution. After this incubation, the sections were washed with PBS and incubated for 1 hour at 37ºC with donkey anti-mouse IgG secondary antibody conjugated to Alexa 555 (Invitrogen) for α-tubulin and donkey anti-rabbit IgG secondary antibody conjugated to Alexa 555 or Alexa 488 (Invitrogen) at a 1:500 dilution for anti-C. glabrata. Afterwards, tissue sections were stained with DAPI (Merck). Then, samples were treated with Autofluorescence Eliminator Reagent (Merck). Finally, sections were observed using a LSM710 confocal laser scanning microscope combined with the upright microscope stand AxioImager.M2 (Zeiss). The spectral system employed was Quasar + 2 PMTs. Zeiss ZEN 2010 Program.
Autofluorescence eliminator reagent
Manufactured by Merck Group
Sourced in United States, Canada
The Autofluorescence Eliminator Reagent is a laboratory product designed to reduce or eliminate autofluorescence in samples. It is intended to be used in various analytical techniques, such as fluorescence microscopy, flow cytometry, and other applications where autofluorescence can interfere with the desired signal.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Lsm 710, by Zeiss (2 mentions)
Fluoromount g, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 or 594 conjugated antibody, by Thermo Fisher Scientific (2 mentions)
Aqueous mounting media, by Thermo Fisher Scientific (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Zen2010 program, by Zeiss (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Axioimager m2 stand, by Zeiss (1 mentions)
Alexa 555, by Thermo Fisher Scientific (1 mentions)
Axio observer, by Zeiss (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Neurotrace 435, by Thermo Fisher Scientific (1 mentions)
Superfrost slide, by Thermo Fisher Scientific (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
Ab125212, by Abcam (1 mentions)
Prolonggold antifade agent with dapi, by Thermo Fisher Scientific (1 mentions)
Alexa conjugated fluorescent secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Lsm 780 confocal microscope, by Zeiss (1 mentions)
20 protocols using autofluorescence eliminator reagent
1
Immunofluorescence Staining of Brain Tissue
2
Immunohistochemical Staining of CNS Tissue
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Standard techniques were used for paraffin embedding and sectioning of CNS tissue. Briefly, paraffin was removed and tissues were rehydrated and pretreated with different buffer solutions. Subsequently, tissue sections were incubated with a primary antibody in PBS-BSA and further incubated with the corresponding secondary antibody conjugated to Alexa 488 (Invitrogen, Carlsbad, CA). Sections were then incubated with a third antibody in PBS and further incubated with the corresponding secondary antibody conjugated to Alexa 555 (Invitrogen). The tissue sections were stained with DAPI and then with autofluorescence eliminator reagent (Merck Millipore). Protocols for immunohistochemical analysis have been described 56 (link). Most of the images were obtained with a Zeiss LSM710 confocal laser scanning microscope equipped with the upright AxioImager.M2 stand (Zeiss), running ZEN 2010 software, or a Zeiss LSM800 confocal laser-scanning microscope equipped with an inverted microscope Axio Observer (Zeiss) and running Zen Blue 2.3 software. Images were deconvoluted using Huygens software (4.2.2 p0) and visualized with ImageJ (NIH).
3
Immunohistochemistry for Hippocampal Sections
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Blocks of approximately 5 mm × 5 mm of the dorsal hippocampus, were embedded in paraffin and cut into 20 µm-thick sections. To reduce autofluorescence, we followed a recently-developed protocol 55, 64 . Briefly, samples were incubated in a 0.5% sodium borohydride (NaBH4; Sigma-Aldrich, 213462) solution, followed by a citrate buffer antigen retrieval (HC-AR) step. Then, sections were microwaved for 10 min in tris buffer saline (TBS) and then incubated for 30 Either the primary or the secondary antibody was omitted in negative-control sections. All sections were counterstained for 10 min with DAPI (Merck; 1:5,000 dilution) to label nuclei.
Immunohistochemistry was followed by a final autofluorescence elimination step, using an autofluorescence Eliminator reagent (EMD Millipore, 2160) and following the manufacturer's instructions. Samples were mounted in VECTASHIELD Antifade Mounting Medium with DAPI (VectorsLab, H-1200) and stored at 4°C until analysis.
Immunohistochemistry was followed by a final autofluorescence elimination step, using an autofluorescence Eliminator reagent (EMD Millipore, 2160) and following the manufacturer's instructions. Samples were mounted in VECTASHIELD Antifade Mounting Medium with DAPI (VectorsLab, H-1200) and stored at 4°C until analysis.
4
Immunohistochemical Analysis of Brain Tissue
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Following perfusion, brains were fixed overnight at 4°C in 4% Paraformaldehyde (PFA) (in 1× phosphate-buffered saline (PBS)) and were then transferred to a 30% sucrose solution (in 1× PBS). Brains were flash frozen in OCT Compound (Tissue Tek) and stored at −80°C until cryosectioning. 50 µm cryosections were mounted onto Superfrost slides (Fisher Scientific) in sets of 3 and allowed to dry overnight at room temperature. Sections were blocked overnight at 4°C in a PBS solution containing 0.3% Triton X-100 and 3% normal donkey serum and then incubated with primary antibody in a PBS-T solution (0.1% Triton X-100 and 1% normal donkey serum), overnight (24 hr at 4°C). The next day, sections were rinsed in PBS-T and incubated in secondary antibody for 2 hr at room temperature in PBS-T along with NeuroTrace-435 (Invitrogen). Sections were treated with an autofluorescence eliminator reagent (EMD Millipore) according to the manufacturer’s guidelines and mounted in Fluoromount-G (Southern Biotech). Details regarding antibodies can be found in the Key Resources Table (KRT).
5
Dual-Labeling Immunofluorescence for Vascular and Immune Markers
6
Immunofluorescent Quantification of Tau Aggregates
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For immunofluorescence, HEK293T cells were washed in PBS and fixed in 4% paraformaldehyde for 10 min. Autofluorescence eliminator reagent (Millipore, Burlington, MA) was added for five minutes and washed with 40% ethanol. Under dark conditions, slides with cells were incubated in 0.0125% Thioflavin S dissolved in 50% ethanol/ PBS for 3 min and washed in 50% ethanol and PBS. Slides were submerged in blocking solution (2% FBS/0.1% Triton-X-100 in PBS) for 30 min. Primary antibody in 2% FBS/PBS was incubated for one hour. After PBS washes, Alexa-fluor 594 conjugated anti-rabbit antibody (Invitrogen, Carlsbad, CA) were added at 1: 500 dilution for one hour. Slides were washed in PBS and placed in 0.5 μg/mg of 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen, Carlsbad, CA) in PBS for 5 min. The coverslips were mounted using Fluoromount-G (Invitrogen, Carlsbad, CA). Fluorescent images were captured with a BZ-X700 Keyence digital microscope (Itasca, Il).
For quantification, different 20X fields of ~20–50 cells were captured for each treatment group using a BZ-X700 Keyence digital microscope (Itasca, Il). Tau positive cells that colocalized with Thioflavin S positive aggregates were calculated as a ratio to total number of tau positive cells and reported as a percentage of Thioflavin S positivity.
For quantification, different 20X fields of ~20–50 cells were captured for each treatment group using a BZ-X700 Keyence digital microscope (Itasca, Il). Tau positive cells that colocalized with Thioflavin S positive aggregates were calculated as a ratio to total number of tau positive cells and reported as a percentage of Thioflavin S positivity.
7
Immunofluorescence Analysis of Myelination Markers
8
Dual Immunofluorescence for GFAP, CD44, and CHI3L1
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Sections were double-labeled with a mouse anti-GFAP or anti-CD44 and a rabbit anti-CHI3L1 antibody overnight. The appropriate secondary antibody was applied [Cy3-donkey anti-rabbit IgG for CHI3L1 and Cy2-donkey anti-mouse IgM for GFAP] and incubated in a 0.1 thioflavin solution (10 min) to visualize aggregated amyloid. Auto-fluorescence was blocked with Auto-fluorescence Eliminator Reagent (Millipore) and sections cover-slipped with aqueous mounting media (Thermo Scientific). Dual immunofluorescence was visualized, and images were acquired using the Revolve Fluorescent Microscope (Echo Laboratories, San Diego, CA, USA) with excitation filters 405, 489, and 555 nm for thioflavin, Cy2, and Cy3, respectively.
9
Astrocytic Tau Pathology in Chronic Traumatic Encephalopathy
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Double stain was used to evaluate whether astrocytes display tau pathology using antibodies to the astrocytic marker GFAP and phosphorylated tau at S396/404, PHF-1, in the hippocampus of CTE stage IV cases [16 (link)]. One section per case was incubated overnight at room temperature with both GFAP (1:1000, DAKO Denmark) and PHF-1 (1:1000, gift from Peter Davies) after 10 min citric acid pH 6 antigen retrieval. Then, the appropriate secondary antibodies were applied for 1 h at room temperature as followed: first Cy5-conjugated donkey anti-mouse IgG for PHF-1 (1:400, Jackson Immuno-research, West Grove, PA) and secondly, after several washes, Cy2-donkey anti-rabbit IgG for GFAP (1:400, Jackson Immuno-research). DAPI, a nuclear fluorescence stain, was employed at 1:2000 at room temperature for 10 min. Auto-fluorescence was blocked with Auto-fluorescence Eliminator Reagent (Millipore, MA, USA) according to manufacturer’s instructions and cover-slipped with aqueous mounting media (Thermo Scientific). Dual immunofluorescence was visualized with the aid of a Revolve Fluorescent Microscope (Echo laboratories, CA, USA) with excitation filters for DAPI (pseudocolored green), Cy2 (emission green; pseudocolored red) and Cy5 (pseudocolored blue) as described previously [17 (link)].
10
Immunohistochemical Analysis of Retinal Proteins
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