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NeuroMab Brain If Protocol 1016

This protocol describes the steps to perform immunofluorescence labeling of free-floating perfusion-fixed brain sections using NeuroMab antibodies. The protocol can be completed in one or two days. It involves section preparation by washing, blocking, primary antibody incubation overnight or for 3 hours, secondary antibody incubation for 1 hour, mounting and coverslipping. Optional steps include Sudan Black B treatment to reduce autofluorescence. Recipes are provided for buffers, vehicle and Sudan Black B solution.

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LucasSánchez
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24 views

NeuroMab Brain If Protocol 1016

This protocol describes the steps to perform immunofluorescence labeling of free-floating perfusion-fixed brain sections using NeuroMab antibodies. The protocol can be completed in one or two days. It involves section preparation by washing, blocking, primary antibody incubation overnight or for 3 hours, secondary antibody incubation for 1 hour, mounting and coverslipping. Optional steps include Sudan Black B treatment to reduce autofluorescence. Recipes are provided for buffers, vehicle and Sudan Black B solution.

Uploaded by

LucasSánchez
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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UC Davis/NIH NeuroMab Facility

Department of Neurobiology, Physiology and Behavior, UC Davis, Davis CA 95616-8519


http://neuromab.ucdavis.edu [email protected] (530) 752-8398

Immunofluorescence Labeling of Free-Floating Perfusion-Fixed Brain Sections

Note: protocol can be completed in one day by shortening primary antibody incubation (Step 3).

Day 1.

1) Section preparation: wash sections four times with ice-cold (4oC) 0.1 M PB (recipe below) with
gentle rocking/agitation at room temperature (RT) for 5 min each.

2) Block: incubate sections in vehicle (recipe below) with gentle rocking/agitation at 4°C for 1 h.

3) Primary antibody incubation: transfer sections into ice-cold (4oC) vehicle containing diluted
NeuroMab(s) and incubate with gentle rocking/agitation at RT for 3 h (or at 4°C overnight).

Note: antibody concentrations and incubation conditions should be determined empirically for each
combination of target sample, NeuroMab, secondary antibody and detection system but, as a
general guide, NeuroMabs should be used between 0.1 and 10 μg/mL.

Day 2.

4) Wash: transfer sections into fresh ice-cold (4oC) vehicle and wash a total of four times with
gentle rocking/agitation at RT for 5 min each.

5) Secondary antibody incubation: transfer sections into ice-cold (4oC) vehicle containing
fluorescently-conjugated anti-mouse secondary antibodies diluted as per manufacturer’s
recommendations (e.g., 0.5 μg/mL of Alexa Fluor 488 conjugated anti-mouse IgG1 antibody,
Thermo Fisher Scientific catalog # A-21121) and incubate with gentle rocking/agitation protected
from light at 4°C for 1 h.

Note: use subclass-specific secondary antibodies matched to the NeuroMab IgG subclass (i.e.,
IgG1, IgG2a or IgG2b) for optimal signal detection and lowest background (Manning et al., PLoS
One 7:e38313, 2012, http://www.ncbi.nlm.nih.gov/pubmed/22675541).

6) Wash: transfer sections into 0.1 M PB, wash twice with gentle rocking/agitation at RT for 5 min
each and wash once with 0.05 M PB with gentle rocking/agitation at RT for 5 min.

7) Mounting: transfer sections into 15 mL Petri dish containing 0.05 M PB, carefully mount onto
gelatin-coated microscope slides and allow to air dry.

8) Sudan Black B treatment (optional, see below).

9) Coverslipping: pipet anti-fade medium (e.g., ProLong Gold Antifade Mountant, Thermo Fisher
Scientific catalog # P36930) on each section according to manufacturer’s instructions, place
A cooperative venture between the University of California at Davis, the National Institutes of Health and Antibodies Incorporated

last updated 14Oct2016 page 1 of 2


UC Davis/NIH NeuroMab Facility
Department of Neurobiology, Physiology and Behavior, UC Davis, Davis CA 95616-8519
http://neuromab.ucdavis.edu [email protected] (530) 752-8398

glass coverslip on top, cure for at least 24 hours and seal edges with nail polish.

Note: tissue autofluorescence can be reduced with an optional Sudan Black B treatment (SBB,
Oliveira et al., Histol Histopathol 25:1017, 2010, https://www.ncbi.nlm.nih.gov/pubmed/20552552).

1) Completely rehydrate slide-mounted sections with ddH20

2) Completely submerge slide in a tray containing 0.05% SBB (recipe below).

3) Incubate with gentle rocking/agitation at RT for 5 min. Sections will become blue.

4) Remove slide from tray and rinse well with ddH20, paying attention to remove any SBB
particulates.

5) Air dry and proceed to coverslipping step.

Recipes:

0.1 M PB: Dilute 500 mL of stock 0.4 M PB (see perfusion protocol for recipe) into ≈1400 mL
ddH20, adjust pH to 7.4 and bring to final volume of 2 L.

Vehicle: 10% normal goat serum and 0.3% Triton X-100 in 0.1 M PB

SBB solution:

1) Prepare stock solution of 1% SBB (Electron Microscopy Sciences catalog # 21610) in 100%
ethanol.
2) Vortex or place into a sonicating water batch for 1 h.
3) Vacuum filter SBB through 0.45 μM cellulose acetate, low protein binding membrane
(Corning catalog # 430314).
4) Prepare working 0.05% SBB solution. Dilute 2.5 mL SBB stock in 47.5 mL 70% ethanol
immediately prior to each use in order to minimize particulates. If autofluorescence levels
are high, then higher SBB concentrations (up to 0.15%) may be justified but increasing SBB
concentrations may also reduce immunofluorescence signal.

Note: stock SBB solution can be reused but must be sonicated for at least 1 h prior to preparing
working solution.

Refs: Oliveira et al., Histol Histopathol 25:1017, 2010 (https://www.ncbi.nlm.nih.gov/pubmed/20552552);


Manning et al., PLoS One 7:e38313, 2012 (https://www.ncbi.nlm.nih.gov/pubmed/22675541);
Bishop et al., J Neurosci 35:14922, 2015 (https://www.ncbi.nlm.nih.gov/pubmed/26538660)

A cooperative venture between the University of California at Davis, the National Institutes of Health and Antibodies Incorporated

last updated 14Oct2016 page 2 of 2

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