At 48 h post-IL-21 and anti-IL-21R antibody injections, mice were deeply anesthetized using isoflurane and euthanized via intracardiac perfusion using 4% paraformaldehyde (Sigma-Aldrich, St Louis, MO) in 100 mM phosphate buffered saline (PBS; pH 7.4, Thermo Fisher Scientific, Waltham, MA) [1 (link)]. Brains were cryoprotected (10–30% sucrose gradient over 2–3 days) and sectioned coronally into 30 µm using a cryostat (Microm HM525, Germany). For each endpoint, 4 representative coronal brain sections of the amygdala and medial prefrontal cortex (mPFC) regions from each of the 4 animals per experimental group were selected at approximately 15 section intervals and stored in PBS. For the immunofluorescence labeling of microglial activation marker CD68, rat anti-mouse CD68 (1:500; AbD Serotec, Hercules, CA) primary antibody was used with Alexa Fluor 594 secondary antibody (1:500). Similarly, for the IL-21R staining with microglia (IBA-1), we use IL21-R (BioXcell, Lebanon, NH) and rabbit anti-IBA-1 (Wako Chemicals, Richmond, VA) antibodies. The secondary antibodies included donkey anti-mouse or anti-rabbit Alexa Fluor 488 or 568. Tissues were then DAPI nuclear counterstained and sealed in slow fade/gold antifade mounting medium (Life Technologies, Carlsbad, CA).
Slow fade gold antifade mounting medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
Slow fade/gold antifade mounting medium is a specialized solution used to preserve and protect fluorescent signals in microscopy samples. It helps maintain the brightness and clarity of fluorescent dyes over an extended period, preventing their premature fading or degradation.
Automatically generated - may contain errors
Lab products found in correlation
Rat anti mouse cd68, by Bio-Rad (2 mentions)
Draq5, by Cell Signaling Technology (2 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Rabbit anti iba1, by Fujifilm (1 mentions)
Il21 r, by BioXCell (1 mentions)
Oct medium, by Thermo Fisher Scientific (1 mentions)
Colorfrost plus slides, by Thermo Fisher Scientific (1 mentions)
Cryostat, by Leica (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Lsm 710 sim, by Zeiss (1 mentions)
Zen 2009 software lsm 710, by Zeiss (1 mentions)
9 protocols using slow fade gold antifade mounting medium
1
Microglia Activation and IL-21R Expression in Brain Regions
2
Harvesting Lymph Nodes for ICP-MS and Immunohistochemistry
3
Immunofluorescence of Microglial Activation
4
Tryptase-Induced Cellular Changes Visualized by Microscopy
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Aliquots of 100 μl/well from 50 × 105 cells/ml suspensions were cultured in 8 chamber polystyrene vessel tissue culture treated glass slides (Falcon, New York, NY) overnight or until confluence. Cells were then left untreated (control) or treated with tryptase (50 nM) for 48 h under culture conditions (37 °C in 5% CO2). Then, supernatants were removed carefully and cells were fixed with 4% paraformaldehyde in PBS for 15 min. One hundred microliters of 50 μg/ml digitonin solution in PBS was added to each individual glass and incubated for 10 min at room temperature, followed by washing three times with TBS-T. The slides were kept in the dark and 50 μl of 1 μg/ml DAPI in TBS/1% BSA was added for 2 min, followed by three times washing with TBS-T. The slides were mounted with SlowFade® gold antifade mounting medium (Life Technologies). Samples were analyzed using a laser-scanning microscope equipped with ZEN 2009 software (LSM 710 SIM; Carl Zeiss, Berlin, Germany).
5
Thioflavin-S Staining for Amyloid
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Sections were rehydrated in an ethanol series (100%, 95%, 70%, 50%) and then incubated in a 0.5% thioflavin-S solution in 50% ethanol for 10 min. Tissues were rinsed twice in 50% ethanol and then rinsed twice in PBS. Sections were mounted and sealed with slow fade/gold antifade mounting medium (Life Technologies, Carlsbad, CA).
6
Immunofluorescence Staining of PA200
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Cytospin slides were prepared from aliquots (100 μL) of cell suspensions and cells were then fixed with 4% paraformaldehyde in PBS for 15 min. An amount of 100 μL of 50 μg/mL digitonin solution in PBS was added to each glass slide and incubated for 10 min at room temperature. Next, 100 μL anti-Psme4/PA200 antibody (1:500) in TBS/1% BSA and/or isotype control at the same concentration were added and left overnight at 4 °C, followed by washing 3 times with TBS-T. Next, Alexa-conjugated secondary antibody diluted (1:1000) in TBS/1% BSA was added and incubated for 1h at room temperature. ActinRed/GreenTM (Molecular Probes) probes were used to stain cells. The slides were kept in the dark, and washed 3 times with TBS-T between each step. Finally, 100 μL of NucblueTM (Molecular Probes) probes in TBS/1% BSA was added for 20 min, followed by 3 times washing with TBS-T. The slides were mounted with SlowFade® gold antifade mounting medium (Life Technologies). Samples were analyzed using a laser-scanning microscope equipped with ZEN 2009 software (LSM 710; Carl Zeiss, Berlin, Germany).
7
Thioflavin S Staining Protocol
8
Immunostaining of 3D and Tissue Spheroids
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Spheroids cultured in Matrigel were fixed for 20 min in 4% paraformaldehyde, washed in 0.5% PBST, permeabilized in 0.4% Triton-X-100/1X PBS for 20 min, and then washed in 0.5% PBST before blocking 1–2 h in 20% donkey serum/PBST. Primary and secondary antibodies were incubated overnight in 3% BSA/PBST. A list of antibodies and dilutions used for immunostaining is provided in S1 Table in S1 Appendix .
Submandibular glands were washed in 0.5% PBST after overnight fixation in 4% paraformaldehyde, permeabilized in 0.4% Triton-X-100/PBS for 30 min and then washed in 0.5% PBST before blocking 1–2 h in 20% donkey serum/PBST. Primary and secondary antibodies were incubated overnight in 3% BSA/PBST. DRAQ5 (Cell Signaling) was used at a dilution of 1:1000 to detect nuclei. Coverslips and slides were mounted using SlowFade® Gold antifade mounting medium (Life Technologies, #P36930).
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9
Immunofluorescence Staining of 3D Spheroids and Submandibular Glands
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Spheroids cultured in Matrigel were fixed for 20 min in 4% paraformaldehyde, washed in 0.5% PBST, permeabilized in 0.4% Triton-X-100/1X PBS for 20 min, and then washed in 0.5% PBST before blocking 1-2 h in 20% donkey serum/PBST. Primary and secondary antibodies were incubated overnight in 3% BSA/PBST. A list of antibodies use for immunostaining is provided in Table S1. Submandibular glands were washed in 0.5% PBST after overnight fixation in 4% paraformaldehyde, permeabilized in 0.4% Triton-X-100/PBS for 30 min and then washed in 0.5% PBST before blocking 1-2 h in 20% donkey serum/PBST. Primary and secondary antibodies were incubated overnight in 3% BSA/PBST. All antibodies and dilutions used for immunofluorescence are listed in Table S2. DRAQ5 (Cell Signaling) was used at a dilution of 1:1000 to detect nuclei. Coverslips and slides were mounted using SlowFade®Gold antifade mounting medium (Life Technologies, #P36930). A list of antibodies use for immunostaining is provided in Table S1.
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