Mouse’s brain was dissected immediately after cervical dislocation euthanasia and fixed by immersion in 4% PFA in PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl2, adjusted to pH 7.2–7.4 with KOH pellets) at 4 °C for 24 h. After that, the brain was washed in large volumes of PHEM buffer, each for 5 min 6 times, to eliminate excess fixatives. The brain was then embedded in a block with a mixture of 0.5% gelatin, 30% bovine serum albumin, and 1% glutaraldehyde (final concentration). Vibratome sections of 60 µm thickness were obtained in a Leica, VT 10000S vibratome. For vessel visualization, brain sections containing the hippocampal head regions were stained as previously described using Isolectin GS-IB4 Alexa Fluor 488 conjugate (Cat#: I21411, ThermoFisher Scientific, Waltham, MA, USA) in 1:100 concentration (Figure 1 c) [48 (link)].
Isolectin gs ib4 alexa fluor 488 conjugate
Manufactured by Thermo Fisher Scientific
Sourced in United States
Isolectin GS-IB4 (Alexa Fluor 488 conjugate) is a fluorescently labeled isolectin derived from the seeds of the plant Griffonia simplicifolia. It binds specifically to alpha-D-galactosyl residues on the surface of cells.
Automatically generated - may contain errors
Lab products found in correlation
Confocal microscope, by Leica (2 mentions)
Prolong diamond antifade, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Matrigel, by BD (1 mentions)
Rhodamine ulex europaeus agglutinin 1, by Vector Laboratories (1 mentions)
Egm 2mv, by Lonza (1 mentions)
Goat anti rabbit conjugated to alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Rabbit anti erg, by Abcam (1 mentions)
Ix70 inverted microscope, by Olympus (1 mentions)
Dp70 digital camera, by Olympus (1 mentions)
7 protocols using isolectin gs ib4 alexa fluor 488 conjugate
1
Visualizing Mouse Brain Vasculature
2
Isolectin-Based Fluorescent Vessel Labeling
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Brain slices were incubated with an IB4 fluorescent probe (Isolectin GS-IB4 Alexa Fluor 488 Conjugate, Thermofisher) in 1:100 concentration with PHM buffer (60 mM PIPES, 25 mM HEPES, 2 mM , adjusted to pH 7.2–7.4 with KOH pellets) and 0.5 mM . Brain slices were incubated over night at 4 C and washed for 5 min with stirring (X6). During incubation, isolectin agglutinates with perivascular cells, probing the vessel wall with a green fluorescent spectrum. Finally, brain slices were mounted in a slide with Prolong Antifade Diamond (ThermoFisher) and were allowed to dry in the dark for 24 h before SLCM imaging.
3
Fluorescence Staining of TLR4 in Retinas
4
Adipocyte Differentiation Assay Protocol
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Matrigel (BD Biosciences); EGM-2 MV (Lonza); Adiponectin human-specific ELISA (Invitrogen KHP0041); Rhodamine Ulex europaeus Agglutinin 1 (Vector labs RL-1062); and Isolectin GS IB4 Alexa Fluor 488 Conjugate (Life Technologies I21411).
5
Epac1 Cdh5 Cre-Lox Mice Eye Analysis
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Three-month-old male and female Epac1 floxed and Epac1 Cdh5 Cre-Lox mice (three in each group) were euthanized with carbon dioxide followed by cervical dislocation. After confirmation of death by cervical dislocation, the eyes were removed and immediately placed in 4% paraformaldehyde in PBS (1X; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) overnight. The following day, the whole globe was transferred to 0.1 M PBS containing 30% sucrose for cryoprotection. Ten-micron sections were cut using a cryostat and stored at 80 °C until use. The sections were rinsed in PBS and put in 5% normal goat serum for 1 h at room temperature for blocking any nonspecific staining, followed by incubation with Isolectin GS-IB4 (Alexa Fluor 488 conjugate, 1:100, Life Technologies) and rabbit anti-epac1 (1:100, Abcam, San Francisco, CA) at 4 °C overnight. After rising in 0.3% Triton/PBS, the sections were incubated with secondary antibody goat anti-rabbit conjugated to Alexa Fluor 594 (1:1,000, Life Technologies) for 2 h at room temperature. The slides were then rinsed in PBS and counterstained with 4',6-diamidino-2-phenylindole (DAPI). The slides were examined with a Leica (Buffalo Grove, IL) confocal microscope.
6
Immunohistochemistry and Immunoblotting Protocols
7
Immunohistochemical Analysis of Brain Endothelial Cells
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Immunohistochemistry staining was performed as previously described by Ara et al., 2011, 2013 [21 (link),22 (link)]. Briefly, coronal brain sections were de-paraffinized in xylene and rehydrated in graded ethanol and distilled water. High temperature antigen retrieval was performed in 10 mM sodium citrate buffer. Sections were blocked and incubated with primary antibodies green fluorescent Alexa Fluor® 488 isolectin GS-IB4 conjugate (an endothelial cell marker, Invitrogen, Carlsbad, CA, USA). The sections were washed in PBS and counterstained with the nuclear dye Hoechst 33258 (2 μg/mL in PBS) for 5 min, and subsequently with cell proliferation marker ki-67. All sections were examined using an Olympus DP-70 digital camera mounted on an Olympus IX70 inverted microscope connected to a computer with Olympus multifunctional software for digital image analysis.
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