Histology

Notes:

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




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:

> December 2012

> November 2012

> October 2012

> September 2012

> August 2012

> July 2012

> June 2012

> May 2012

> April 2012

> Feb 2012

> Jan 2012; 2011

Notes by Antigen:

[Calbindin]

[Calretinin]

[Parvalbumin]

[Somatostatin]

[Neuropeptide Y]

Division of Science Summer Undergrad Poster Session 2012 [.pdf]

comparison of inhibitory markers: [.jpg] [.psd]

0910-12: [DS Laminar Development]

April 2012

March 2012

February 2012

January 2012

December 2011

September 2011

August 2011

July 2011

June 2011

May 2011

April 2011

March 2011

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 1209

Brain 1256

Brain 1257

Brain 1258

Brain 1160 lesion #1: 1241 #2: 283

SMI-32 & NeuN Staining Summer/Fall 2016

Brain 1223

Julie Miller's Old Stuff

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):

[GCaMP/YFP]

[fSST]



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



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.