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DELLA CROCE DEARNALEY, Anna

14/12/16

Immunohistochemical detection of Glial Acidic Fibrillary Protein (GFAP) in Mouse


Brain
Monday, October 31st 10am 12pm (Dr. Hobbs)
Wednesday, November 30th 11am- 3pm (Dr. Hobbs)
Introduction:
Immunohistochemistry takes its name from "immuno", referring to antibodies used in
the procedure, and "histo," meaning tissue. Indirect Immunohistochemistry refers to the
process of detecting and amplifying antigens in cells of a tissue section by exploiting the
principle of antibodies binding specifically to antigens in biological tissues. Glial Fibrillary
Acidic Protein (GFAP) is an intermediate filament protein expressed by numerous cells in the
central nervous system that can be used to distinguish astrocytes from other neuroglial cells.
The purpose of this practical was to recognize the distribution of GFAP in sections of a
paraffin embedded sagittal section of an adult mouse brain and to visualize the distribution
and functions of astrocytes within the brain.
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Materials and Methods:
The protocol attached in the appendix was closely followed throughout the practical and
the follow-up. However, a few changes were made to the GFAP staining of the mouse brain.
Step 11 in the original protocol was not carried out as stated. This was due to the risk of the
mouse brain on the section of the slide drying out or being damaged. As a result, the slide was
placed in Scotts tap water for two minutes and then the slide was immediately placed in the
Coplin jar with a solution of 70% alcohol.
Results:
The practical was designed to locate the protein GFAP in the sagittal section of mouse
brain through immunohistological staining. This then allowed the astrocytes expressing
GFAP to be identified and observed. The expectation was that immunohistological staining
would occur and would cause a brown DAB precipitate to result where the GFAP protein was
located. The results of the practical varied from this hypothesis as no immunohistological
staining took place and the only staining that could be successfully noted was that originating
from the counterstain. The counterstain allowed the form of the sagittal mouse brain to be
observed and some features could also be noted such as the hippocampus, the cerebellum and
corpus callosum. As a result of the failure of the immunohistological staining, GFAP cannot

be located thus, the astrocytes present in the section cannot be identified and their distribution
in the sagittal mouse brain section cannot be observed.

Conclusion:
As predicted from the results of this practical, features of the sagittal mouse brain can be
identified such as the hippocampus, the cerebellum and the corpus callosum. This meant it
was possible to estimate the location of other sections and features of the mouse brain. As
previously mentioned in the result section of this report, the fibrous astrocytes could not be
observed and therefore GFAP could not be located. The protoplasmic astrocytes present in the
section of adult sagittal mouse brain have a lower expression of GFAP and therefore could
not be observed.

Appendix
Materials
Youhavebeenprovidedwithaslidecontainingasagittalsectionofanadult
mousebrain.Itisaparaffinembeddedsectionthathasalreadybeendewaxedand
rehydrated.ItisprovidedinTrisBufferedSaline(TBS)inaCoplinjar.
Solutions
Note:onlypartialinformationisgivenonthecompositionofthesolutions.Itisnot
necessarytoknowdetailsoftheconstituents
Intubes:
1.Redtube:Blockingsolution(2%BovineserumalbumininTBS)
2.Yellowtube:Primaryantibody(RabbitantiGFAPIgG,predilutedto1:500in
TBS)
3.Greentube:Horseradishperoxidase(HRP)conjugatedsecondaryantibody
(SwineantirabbitIgG,conjugatedtoHRPandpredilutedto1:100inTBS)
4.Cleartube:Diaminobenzidine(DAB)solution(DABdilutedinTrisand
includingH2O2asasubstrate)
InCoplinjars:
1.TrisBufferedSaline(TBS)
2.Scottstapwater

3.70%Alcohol
4.90%Alcohol
5.Absolutealcohol(100%ethanol)
6.Histoclear
Other:
1.SmallbottleofGillsHaematoxylin
2.SmallbottleofDPX
Protocol
Pleaseensurethatyoustainthesideoftheslidewiththetissueonit!
1.Placeyourslideontherackontheplastictrayandusingadisposablepipette,
addsufficientBlockingsolution(Redtube)justtocoverthesectionentirely.Leave
for3minutes.
2.Drainoffthesolutionintotheplastictray.Thesectionisinthemiddleofacircle
thathasbeenmarkedoutwithapenthatgeneratesawaterresistantfilm.This
meansthatasolutioncanbeappliedwithinthisringwithoutspreadingouttothe
restoftheslide.Thisisimportantwhenapplyingexpensiveortoxicsolutionsto
keepthevolumesusedtoaminimum.Thususingadisposablepipette,coverentire
section(notentireslide!)withinthewaterresistantringwiththerabbitantiGFAP
antibody(Yellowtube).Leavefor30minutes.
3.DrainoffthesolutionintotheplastictrayandplacetheslideinTBS(inthe
Coplinjarthatyouoriginallytooktheslidefrom)for3minutes.
4.DrainofftheTBSintotheplastictrayandreplacetheslideontherack.Forthis
andallsubsequentstepsusingthesliderack,makesurethesectionisuppermoston
theslide!Usingadisposablepipette,placejustenoughoftheconjugated
secondaryantibody(Greentube)ontheslidetocovertheentiresection(seenote2
aboveaboutapplyingsolutionswithinthewaterresistantring).Leavefor30
minutes.
5.WashagainbyreturningtheslidetotheCoplinjarcontainingTBSfor5
minutes.

6.DrainofftheTBSintotheplastictrayputtheslidebackontherack.Usinga
disposablepipette,covertheentiresectionwiththeDABreagent(Cleartube).
Again,seenote2aboveaboutapplyingsolutionswithinthewaterresistantring.
RememberthatDABisverytoxicandsodothisstepCAREFULLY.Leavefor10
minutes.
7.HoldtheslidewiththeforcepsprovidedanddraintheDABreagentbytapping
theslideonto3or4sheetsofthetissuesprovided.Again,performthisstep
carefullyandensurethatthesmallamountofwasteDABisconfinedtothetissues.
PlacetheslideintothejarcontainingTBSandleavefor1minute.
8.DrainofftheTBSintotheplastictrayandputtheslidebackonthesliderack.
UsingadisposablepipetteaddenoughGillshaematoxylinfortocoverthesection.
Leavefor30seconds.
9.DrainoffthehaematoxylinintotheplastictrayandplaceslideintheCoplinjar
containingtheTBS.Usingtheforceps,lifttheslideupanddownintotheCoplin
jar3timestohelprinseoffthehaematoxylinandthenleaveitintheTBSfor1
minute.
10.DrainofftheTBSintotheplastictrayandplacetheslideintotheCoplinjar
containingScottstapwaterfor2minutes.
11.Withtheslidelabelfacingtheblottingpaper,gentlylowertheslideandpress
thebackoftheslidetoensurethatallwaterisabsorbed.Dothissmoothlyanddo
NOTmovetheslideinanydirectiononceitisincontactwiththeblottingpaperor
youmayloseordamagethesection.Simplygentlylowertheslide,pressonthe
backofitandgentlyliftstraightupagain.
12.Wipethebackoftheslidedrywithatissue,takingcarenottotouchthesection
ontheotherside.PlaceintheCoplinjarcontaining70%alcohol.Leavefor2
minutes.
13.Draintheexcessalcoholintotheplastictrayandthenplacetheslidein90%
alcohol.Leavefor2Minutes.
14.Draintheexcessalcoholintotheplastictrayandthenplacetheslidein
absolutealcohol.Leavefor2Minutes.
15.Holdingtheslidevertically,brieflydrainbyplacingtheendoftheslideaway
fromthelabelontothesurfaceoftheblottingpapersothatonlytheedgeofthe
slideistouchingandtheabsolutealcoholisdrainingdowntheslideontothe
blottingpaper.ThenplacetheslideintheCoplinjarcontainingHistoclearand

leavefor2minutes.Agitateevery30secondsbyraisingandloweringtheslidein
theCoplinjarabout5times.
16.Checkyourslide.Ifthesectiononithasawhiteemulsion/film,thengobackto
stage14andcontinuefromthere.
17.ApplyoneortwosmalldropsofDPXmountingmediatothesection.Transfer
theDPXusingthesmallwoodenstickprovided.TheDPXwillclingtothestick
andthenfalltotheslide.Donottouchthesectionwiththestick.Carefullylaya
coverslipontheslide,coveringthesection.Oncethecoverslipisinplace,donot
moveitasyoumaydamagethesection.
TheDPXonthesectionwillbelefttopolymeriseandyouwillbeviewingyour
ownsectionsinthepractical:Immunohistochemistry:followup.

School of Bioscience Education, Kings College London


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