Histology
Please keep all areas of the histology hall clean. If a solution is running low, please alert Víkko (suarez@brandeis.edu).
Please log all brains on the follow form: https://docs.google.com/spreadsheets/d/1Hnk-VXwZ7fK3y3SbjfIfBfXe9gqagCBkhs6_0Kff9Qc/edit?usp=sharing
This will help us declutter the fridge space and keep track of brains that are sitting around for a longer time.
Please label all brains with the following information:
Initials
Ferret Number/Shave Pattern
Perfusion Date
Date Transferred to ___ (PFA-> 10% Sucrose -> 30% Sucrose).
A comprehensive list of histological reagents can be found here: https://docs.google.com/spreadsheets/d/1xWuZ0Rg9_VfMdmPUnUmchtaUjjuy2VBV7KWbI2zfDE4/edit#gid=0
Here are general reminders:
Antibodies should be stored in their appropriate temperatures (-20 C vs 4 C) and aliquoted to avoid excessive freeze/thaw cycles.
Aliquoting minimizes damage due to freezing and thawing; Aliquots should be frozen and thawed once, with any remainder kept at 4 C.
Upon receiving an antibody, recommendation is to centrifuge at 10,000 g for 20 seconds and proceed to aliquoting in microcentrifuge tubes.
The smallest aliquot recommendation is 10 uL but please consult the manufacturer for the most updated recommendations.
Brains should be logged in the Brain Database.
Brains should be processed (sliced, mounted, etc) within a month of their extraction to avoid a back-log.
I would also like to point people to the Abcam Antibody Storage guide which is a pretty useful resource; every manufacturer has their own guide but I am partial to Abcam (they are in Waltham and work!)
Protocols, Manuals, and Solutions
PERFUSION SOLUTION RECIPE USING 10% FORMALIN
***Make this under the hood***
In a 500 mL flask, mix:
50 mL 10% Formalin solution (found under hood)’
250 mL 0.2M phosphate buffer
200 mL dH20
*make this solution the day you plan to perfuse, and dispose of any excess in the waste container under the hood. Do not store.
If 10% Formalin is not available:
PERFUSION SOLUTION RECIPE USING PARAFORM
In a 500 mL flask:
Heat 220 mL dH20 to 60 degrees C
Add 20 g paraform (weigh out under hood; wear mask)
Stir for 10 minutes
Add NaOH while stirring (~1 pellet). Solution should turn mostly clear.
Add 30 mL dH2O
Add 250 mL 0.2M phosphate buffer
AFTER PERFUSION:
Brain is typically removed and stored in a glass jar that is filled with enough 4% PFA to cover the brain. If the brain will be processed for histology, do not leave in PFA for longer than 24 hours (move into a jar of PBS at that point). If histology won’t be done for a while, leave in PB until a week before histology will be performed.
Within a week before performing histology, move the brain into a 10% sucrose solution followed by a 30% sucrose solution (made in PB), waiting at each step until the brain sinks (normally 1-2 days)
NOTE: Brains should be processed within 2 weeks of experiment, ideally.
STOCK SOLN RECIPES:
*Note: MilliQ is kept in a container on the top shelf at the back of the Histology Hall (Bassine 327). If this container is empty, you can fill the container in the Katz lab or the Transgenic Facility on the 2nd floor of Bassine.
0.2M SODIUM PHOSPHATE MONOBASIC STOCK
13.8g Monobasic
500 mL MilliQ (if unable to get MilliQ, dH2O ok but MilliQ is better)
0.2M SODIUM PHOSPHATE DIBASIC STOCK
26.8g Dibasic
500 mL MilliQ
0.1M PB (1000 mL), pH 7.4
95 mL Monobasic
405 mL Dibasic
500 mL MilliQ
0.1M PBS (1000 mL), pH 7.4
95 mL Monobasic
405 mL Dibasic
500 mL MilliQ
9 g Sodium Chloride
MAKING 0.1M PBS FROM 0.2M PB
Measure 50% 0.2M PB (50% total volume)
In another container, measure 50% MilliQ
Add 1.8% saline (sodium chloride) to MilliQ
(1.8g*totalvolume/volumeMilliQ)
Once dissolved, add the 2 solutions together, test pH for accuracy
ex) for 100 mL total soln 0.1 M PBS
In one jar: 50 mL 0.2M PB
Another jar: 50 mL MilliQ
Add 1.8% saline:
1.8g*100mL/50mL = 0.9g sodium chloride
*If any of the stock solutions are running low, replenish them or email the drug Czar (currently Vikko Suarez: suarez@brandeis.edu
Protocols
Slicing using Sliding Microtome
Confocal Imaging:
- imaging Interneurons with z-Series
(and drawing ROIs)
IHC Manual
Slicing using Sliding Microtome
Confocal Imaging:
- imaging Interneurons with z-Series
(and drawing ROIs)
Recommended reading for individuals starting IHC:
Histological-based Stainings using Free-Floating Tissue (video coming soon!)
Useful Stuff
DAKO Immunochemical Staining Methods
Immunostar Immunochemistry Instruction Manual
Leica Microtome Manual (also under "Manuals" in "Devices" menu)
Recommended reading for individuals starting IHC:
Histological-based Stainings using Free-Floating Tissue (video coming soon!)
Recommended Readings
Recommended reading for individuals starting IHC:
Histological-based Stainings using Free-Floating Tissue (video coming soon!)
Abcam's Introduction to Immunohistochemistry
Inhibitory Interneuron Project (Julie Miller's Old Stuff) :
i: imaged; R: ROIs taken; cc: cell count done
[iteration 8]: December 2012
[iteration 7]: December 2012 i
[iteration 6]: December 2012 i
[iteration 5]: December 2012 i
[iteration 4]: November - December 2012 i, R
[iteration 3]: November 2012 i
[iteration 2]: August 2012 i
[iteration 1]: July 2012 i, R, cc
Notes by Month:
> May 2012
> Feb 2012
Notes by Antigen:
Division of Science Summer Undergrad Poster Session 2012 [.pdf]
comparison of inhibitory markers: [.jpg] [.psd]
0910-12: [DS Laminar Development]
Nissl Staining Summer/Fall 2016
Brain 1144 lesion #1: 1316 #2: 256
Brain 1148 lesion #1: 1118 #2: 314
Brain 1149 lesion #1: 1036 #2: 274
Brain 1160 lesion #1: 1241 #2: 283
SMI-32 & NeuN Staining Summer/Fall 2016
Old Stuff from Julie Miller -
IHC Records and Imaging
Images taken on Leica SP2 confocal microscope, 10x zoom
[settings]
1011-02:
Virus (notes by brain ID):
Information about the Keyence
Keyence:
Keyence BX-Z 710 is a Fluorescence Microscope that supports brightfield, phase contrast, oblique illumination, and fluorescence observation all within a single compact unit.
The Keyence scope has 3 channels (DAPI, GFP, and Texas Red). If you want to use it to image your brain slices, ensure that your fluorophores are within those working ranges. (Image taken from ThermoFisher.com - Fluorescence SpectraView
Information about the Zeiss
Zeiss LSM 880:
LSM880 2+1Ch: confocal microscope with 7 laser lines (405/458/488/514/561/594/633nm) and 3 detectors of which 1 a GaAsP detector (for green-yellow detection, where GaAsP has highest sensitivity, however, you can also direct other spectra to this detector.
