Answer:
o TC Supernatant ~1:2 – 1:5 dilution for IB, IP, and IF staining 
~1:5 – 1:20 dilution for immunoperoxidase-based staining 
o Purified 1-10µg/ml for IB, IP, and IF staining 
0.1-1µg/ml immunoperoxidase-based staining 

Answer:
~1mg/ml

Answer:
The Material Transfer Agreement/Compliance Statement is a required document. This information ensures that these research tools are not being used for production purposes and enable the NIH to track the end-users of these antibodies . An MTA/Compliance Statement MUST BE ON FILE before your first order of NeuroMabs are shipped to you. An MTA need not be completed for every order. 
A blank MTA may be downloaded from the NeuroMab website - www.neuromab.org

Answer:
1) Rinse cells cultured on cover slips 3X with ice-cold PBS/Ca/Mg (10 mM Phosphate buffer, pH 7.4; 
0.15 M NaCl, 1 mM MgCl2/ 1 mM CaCl2). 
2) Add freshly prepared ice-cold 3% paraformaldehyde/0.1% Triton X-100/PBS/Ca/Mg. Incubate at 
4°C for 30 min with gentle rocking/agitation. 
3) Rinse 3 times in PBS + 0.1% Triton X-100 at RT. 
4) Incubate 30-45 min at RT with gentle rocking/agitation in blotto TBS-T (4% nonfat milk powder in 
10 mM Tris, pH 8.0/0.15 M NaCl + 0.1% Triton X-100). 
5) Add NeuroMab diluted in blotto-TBS-T. NeuroMab dilutions will have to be determined empirically 
for each combination of target sample and NeuroMab, but as a general guide, NeuroMab tissue 
culture supernatants should be used at 1:2-1:10, purified NeuroMab IgG's from 1-10 µg/ml. 
Incubate 45 min- 1 hour at RT with gentle rocking/agitation. 
6) Wash 3 times 5 min each in blotto TBS-T at RT with gentle rocking/agitation. 
7) Incubate in fluorescent second antibody diluted in at RT with gentle rocking/agitation 45 min-1 
hour at RT with gentle rocking/agitation. 
8) Wash 3X 5 min each in PBS+ 0.1% TX-100 at RT with gentle rocking/agitation. 
9) Mount on microscope slides in anti-fade mounting medium, seal with nail polish. View under 
epifluorescence. 
 
from the NeuroMab website.

Answer:
1) Weigh two adult rat brains, should be ≈ 4-5 grams total. 
2) Place in 40 ml of ice-cold homogenization buffer (0.32 M sucrose, 5 mM Na Phosphate buffer pH 
7.4, 0.1 M Na fluoride, with fresh addition of 2 µg/ml aprotinin, 1 µg/ml leupeptin, 2 µg/ml antipain, 
10 µg/ml benzamidine and 0.5 mM PMSF) in a 50 ml Potter-Elvehjem Tissue Grinders 
3) Homogenize on ice using 10 strokes of a Teflon-glass homogenizer attached to a 3/8” variable 
speed drill at 2,500 rpm. 
4) Centrifuge homogenate in Sorvall SS-34 rotor or equivalent at 750 X g, 4°C X 10 min. 
5) Collect the supernatant and save (this has brain membranes = crude synaptosomes) 
6) Recover additional membranes from pellet by scraping off top, lightly colored layer (do not disturb 
deeper layer of red blood cells). Save pellet as “low speed pellet” or LSP. 
7) Rehomogenize pellet using 40 ml of buffer, this time use only 8 strokes. 
8) Centrifuge this homogenate in Sorvall SS-34 rotor or equivalent at 750 X g, 4°C X 10 min. 
9) Collect supernatant and combine with supernatant from first homogenization. 
10) Centrifuge pooled supernatants in Sorvall SS-34 rotor or equivalent at 40,000 X g, 4°C for 90 
min (or ultracentrifuge at 100,000 X g for one hour if available). 
11) Save pellets, these are the crude membrane fraction. Resuspend in 2.5 ml for every gram of 
brain used. 
12) Rehomogenize using hand held Dounce glass-glass homogenizer. Should yield ≈10-15 mg 
protein/ml. 
Can modify protocol to brains from any mammalian species by proportionally adjusting volumes. 
 
from the NeuroMab website

Answer:
Day 1. 
1) Wash sections 4X with gentle rocking/agitation at 4ºC, 5 min each in ice-cold 0.1 M PB (0.1M Na 
Phosphate pH 7.4). 
2) Block sections in vehicle (10% normal goat serum in 0.1 M PB+ 0.3% Triton X-100) for 1 h with 
gentle rocking/agitation at 4ºC 
3) Incubate sections in NeuroMab diluted in ice-cold vehicle. Dilution factors for NeuroMabs should 
be determined empirically, but generally NeuroMab tissue culture supernatants should be used at 
1:2-1:10, purified NeuroMab IgG from 1-20 µg/ml. Incubate sections overnight at 4ºC with gentle 
rocking/agitation. 
Day 2. 
4) Wash sections 4X with gentle rocking/agitation at 4ºC, 5 min each in ice-cold vehicle. 
5) Incubate sections in appropriate fluorescent goat anti-mouse secondary (e.g., Alexa Fluor 488 
goat anti-mouse IgG Invitrogen A11001 at 1:2000) for 1 h with gentle rocking/agitation at 4ºC. 
From this step on the sections should be protected from room light. 
6) Wash sections with gentle rocking/agitation at 4ºC, 5 min in vehicle. 
7) Wash sections with gentle rocking/agitation at 4ºC, 5 min in 0.1 M PB. 
8) Wash sections with gentle rocking/agitation at 4ºC, 5 min in 0.05 M PB. 
9) Mount on gelatin-coated microscope slides (floating in 0.05 M PB in a 15 ml Petri dish). 
10) Air dry 
11) Put 15 µl mounting media (e.g., Prolong Gold antifade reagent, Invitrogen P36930) on each 
section and coverslip. 
12) Seal with nail polish. 
 
from the NeuroMab website.

Answer:
Day 1. 
1. Wash sections 2X with gentle rocking/agitation at 4ºC, 5 min each in ice-cold TBS (0.15M NaCl, 
50 mM Tris, pH 7.5). Use 6-well plate with net insert for accelerated washing. One well fits at most 
10 sections. Do not overflood the well with TBS since floating sections may be caught at rims of the 
inserts. Be gentle lifting and lowering the inserts while transferring to prevent sections from 
breaking due to surface tension of the buffer. 
2. Permeabilize sections in 0.5% TX-100 in TBS for 40-45 minutes at 4ºC with gentle 
rocking/agitation. 
3. Wash sections 2X, 5 min each in ice-cold TBS with gentle rocking/agitation. 
4. Block sections in ice-cold vehicle (5% normal horse serum, 0.1% TX-100 in TBS) for 45 minutes 
at 4ºC with gentle rocking/agitation. 
5. Incubate sections in NeuroMab diluted in ice-cold vehicle. Dilution factors for NeuroMabs should 
be determined empirically, but generally NeuroMab tissue culture supernatants should be used at 
1:2-1:20, purified NeuroMab IgG from 100 ng/ml to 1 µg/ml. Incubate sections overnight at 4ºC with 
gentle rocking/agitation. 
Day 2. 
6. Wash sections 5X, 5 min each in ice-cold TBS with gentle rocking/agitation. 
7. Incubate in ice-cold biotinylated secondary antibody (horse anti-mouse IgG, Vector Labs BA- 
2001, at 1:250 in vehicle) for 1 hour at 4ºC with gentle rocking/agitation. 
8. Just before the end of the secondary antibody incubation, make ABC reagent (Vectastain Elite 
Peroxidase System, Vector Labs PK-6100). For each 5 ml of TBS (NO serum), add 2 drops of ABC 
kit Solution A and 2 drops of Solution B. The ABC solution requires a 30 minute pre-incubation 
period at 4ºC to work properly. 
9. While the ABC solution is being activated, wash section 5X, 5 minutes each in ice-cold TBS with 
gentle rocking/agitation. 
10. Incubate sections in ice-cold ABC solution for one hour at 4ºC with gentle rocking/agitation. 
11. Wash sections 3X, 5 min each in TBS with gentle rocking/agitation. 
12. Wash sections 2X, 5 min each in TB (50 mM Tris, pH 7.5) with gentle rocking/agitation. While 
washing, prepare DAB/NAS developing solution (see below). 
13. Develop sections in DAB/NAS solution. Developing duration will vary so visually monitor 
intensity of reaction constantly so as to avoid over developing. The optimal development time 
should be determined empirically for each NeuroMab. 
14. To stop developing, transfer developed sections to TB and wash 2 more times with TB. 
15. Mount sections on gelatin-subbed slides: Use 15 cm Petri dish filled with TB, submerge a slide 
in the buffer. Using a brush, position a section right above the desired mounting area and while 
holding the section with the brush, gently lift the slide out of the TB. Tilt the slide to remove 
excessive buffer and let it air dry for a couple of hours (overnight is better), at RT. 
16. Dehydrate mounted sections successively in 70%, 95%, 100% (2X), Citrus Clearing Solvent 
(Thermo Scientific Richard-Allan Citrus Clearing Solvent 8301) (2X), 5 min each under a fume hood. 
Coverslip with Electron Microscopy Sciences DPX mountant (13510). The slides should lay flat in 
fume hood for 3-5 days. 
DAB/NAS solution (for 100 ml) 
0.04 g DAB (3-3’ diaminobenzidine tetrahydrochloride) 
0.3 g NAS (nickel ammonium sulfate) 
Dissolve in 100 ml of TB and filter through a Whatman #2 filter. 
Immediately before developing, add 10 µl of 30% H2O2. 
*prepare immediately before use. 
*DAB is a suspected carcinogen. Wear protection gears and work under a hood when 
handling dry DAB powder. 
*all the glassware used during preparation of DAB solution should be cleaned with bleach. 
 
from the NeuroMab website.

Answer:
Not currently. Hopefully, we will be able to offer these in the near future. Please keep checking the website for availability.

Answer:
1. Transfer proteins from SDS gel to pure nitrocellulose membrane (0.45 µm pore size, e.g. BioRad 
Transblot 162-0115) 
2. Place blot(s) in 4% non-fat dry milk (blotto*) in 20 mM Tris-HCl, pH 8.0/ 0.15 M NaCl (TBS). Final 
pH should be ≈ 7.5. Incubate at RT for 45 min with gentle rocking/agitation. 
3. Dilute NeuroMab in TBS-Blotto. NeuroMab dilutions will have to be determined empirically for 
each combination of target sample, NeuroMab and secondary antibody/detection system, but as a 
general guide, NeuroMab tissue culture supernatants should be used at 1:2-1:20, purified 
NeuroMab IgG's from 100 ng/ml to 1 µg/ml. Place blots in NeuroMab solution, incubate from 45 min 
at RT (or overnight at 4°C) with gentle rocking/agitation. These incubation conditions can also vary 
with amount of target antigen on blot, and secondary antibody/detection system. Usually, 45 min at 
RT is sufficient, but some target sample, NeuroMab and secondary antibody/detection system 
combinations will need more extensive incubations. 
4. After incubation, remove NeuroMab solution (can save at 4°C for repeated reuse after adding 
sodium azide to 10 mM final) and add a small volume of TBS-Blotto to incubation chamber to rinse 
out excess antibody. Then add TBS-Blotto, and incubate with gentle rocking/agitation for 10 min. 
Repeat wash 2X. 
5. Add blots to anti-mouse secondary antibody solution. (e.g. Antibodies Inc. 48-146-H, horseradish 
horseradish peroxidase conjugated goat anti-mouse IgG at 1:2000), diluted in TBS-Blotto. Incubate 
45 min at RT with gentle rocking/agitation. Can also save and re-use secondary antibody. 
6. Rinse blots in TBS or PBS, pH 7.5. Wash 3X 10 min each with gentle rocking/agitation. 
7. Develop as per guidelines for your specific detection system (e.g. Renaissance ECL reagent 
from NEN/Dupont). 
 
from the NeuroMab website.