Immunohistochemistry > Background Section
The Core will provide a selected array of immunohistochemistry stains for validated and tested antibodies. Unstained sections can also be provided to the investigator for special studies.
Optimal immunostaining requires good tissue fixation and high quality antibodies. Formalin has been widely used, particularly in clinical investigation. However, some antibodies do not work in formalin-fixed kidneys. For these studies antigen retrieval is necessary. The Core Investigators have long experience in investigations of the effect of kidney fixation on immunostaining and have developed a practical protocol to determine the fixative(s) that are best suited for a specific antibody in kidney immunostaining. We have found that in most cases, kidney sections fixed with one of the following 4 fixatives will work well for most commercially available or investigator-generated antibodies: FPAS (3.7% formaldehyde, 10 mM sodium m-periodate, 40 mM phosphate buffer, and 1% acetic acid); GPAS (2.5% glutaraldehyde, 10 mM sodium m-periodate, 40 mM phosphate buffer, and 1% acetic acid); F (formalin); and PFA (4% paraformaldehyde). For example, cyclooxygenase-2 antibody (Cayman Chemicals) works well in kidney fixed with FPAS and GPAS, but does not work in kidneys fixed with PF (15, 16, 17). 5-tryptophan hydroxylase antibody works well in formalin fixed kidneys, but does not work in FPAS or GPAS fixed kidneys. Anti-renin antibody works well in kidney fixed with FPAS- or PAS- or F, but not when kidney tissue is fixed with PF (18), (19). In the TMA block, mouse and rat kidney cortex tissue sections fixed with each of these four fixatives are included.
Below is an example of renal tubular immunohistochemistry stain:
Publications for Immunohistochemistry (5)
Cheng HF, Wang JL, Zhang MZ, Wang SW, McKanna JA, Harris RC. Genetic deletion of COX-2 prevents increased renin expression in response
to ACE inhibition. Am J Physiol Renal Physiol (2001) 280:F449-56
View abstract View in PubMed
Cyclooxygenase-2 (COX-2) is expressed in macula densa (MD) and surrounding
cortical thick ascending limb of the loop of Henle (cTALH) and is involved
in regulation of renin production. We and others have previously found
that selective COX-2 inhibitors can inhibit renal renin production (Cheng
HF, Wang JL, Zhang MZ, Miyazaki Y, Ichikawa I, McKanna JA, and Harris RC.
J Clin Invest 103: 953-961, 1999; Harding P, Sigmon DH, Alfie ME, Huang
PL, Fishman MC, Beierwaltes WH, and Carretero OA. Hypertension 29:
297-302, 1997; Traynor TR, Smart A, Briggs JP, and Schnermann J. Am J
Physiol Renal Physiol 277: F706-F710, 1999; Wang JL, Cheng HF, and Harris
RC. Hypertension 34: 96-101, 1999). In the present studies, we utilized
mice with genetic deletions of the COX-2 gene in order to investigate
further the potential role of COX-2 in mediation of the renin-angiotensin
system (RAS). Age-matched wild-type (+/+), heterozygotes (+/-), and
homozygous null mice (-/-) were administered the angiotensin-converting
enzyme inhibitor (ACEI), captopril, for 7 days. ACEI failed to
significantly increase plasma renin activity, renal renin mRNA expression,
and renal renin activity in (-/-) mice. ACEI increased the number of cells
expressing immunoreactive renin in the (+/+) mice both by inducing more
juxtaglomerular cells to express immunoreactive renin and by recruiting
additional renin-expressing cells in the more proximal afferent arteriole.
In contrast, there was minimal recruitment of renin-expressing cells in
the more proximal afferent arteriole of the -/mice. In summary, these
results indicate that ACEI-mediated increases in renal renin production
were defective in COX-2 knockout (K/O) mice and provide further indication
that MD COX-2 is an important mediator of the renin-angiotensin system.
Zhang MZ, Harris RC, McKanna JA. Regulation of cyclooxygenase-2 (COX-2) in rat renal cortex by adrenal
glucocorticoids and mineralocorticoids. Proc Natl Acad Sci U S A (1999) 96:15280-5
View abstract View in PubMed
Production of prostaglandins involved in renal salt and water homeostasis
is modulated by regulated expression of the inducible form of
cyclooxygenase-2 (COX-2) at restricted sites in the rat renal cortex.
Because inflammatory COX-2 is suppressed by glucocorticoids, and
prostaglandin levels in the kidney are sensitive to steroids, the
sensitivity of COX expression to adrenalectomy (ADX) was investigated. By
2 weeks after ADX in mature rats, cortical COX-2 immunoreactivity
increased 10-fold in the cortical thick ascending limb and macula densa.
The constitutive isoform, COX-1, was unchanged. The magnitude of the
changes and specificity of COX-2 immunoreactivity were validated by in
situ hybridization histochemistry of COX-2 mRNA and Western blot analysis.
Increased COX-2 activity (>5-fold) was documented by using a specific
COX-2 inhibitor. The COX-2 up-regulation in ADX rats was reversed by
replacement therapy with either corticosterone or deoxycorticosterone
acetate. In normal rats, inhibition of glucocorticoid receptors with RU486
or mineralocorticoid receptors with spironolactone caused up-regulation of
renal cortical COX-2. These results indicate that COX-2 expression in situ
is tonically inhibited by adrenal steroids, and COX-2 is regulated by
mineralocorticoids as well as glucocorticoids.
Cheng HF, Wang JL, Zhang MZ, Miyazaki Y, Ichikawa I, McKanna JA, Harris RC. Angiotensin II attenuates renal cortical cyclooxygenase-2 expression. J Clin Invest (1999) 103:953-61
View abstract View in PubMed
We have previously shown that in rat renal cortex, cyclooxygenase-2
(COX-2) expression is localized to cTALH cells in the region of the macula
densa, and that dietary salt restriction increases COX-2 expression.
Administration of the angiotensin converting inhibitor, captopril, further
increased COX-2 mRNA and renal cortical COX-2 immunoreactivity, with the
most pronounced expression in the macula densa. Administration of an AT1
receptor antagonist, losartan, also significantly increased cortical COX-2
mRNA expression and COX-2 immunoreactivity. Mutant mice homozygous for
both Agtr1a and Agtr1b null mutations (Agtr1a-/-,Agtr1b-/-) demonstrated
large increases in immunoreactive COX-2 expression inthe cTALH/macula
densa. To determine whether increased COX-2expression in response to ACE
inhibition mediated increases in renin production, rats were treated with
captopril for one week with or without the specific COX-2 inhibitor,
SC58236. Plasma renin activity increased significantly in the captropril
group, and this increase was significantly inhibited by simultaneous
treatment with SC58236. Thus, these studies indicated that angiotensin II
inhibitors augment upregulation of renal cortical COX-2 in states of
volume depletion, suggesting that negative feedback by the
renin-angiotensin system modulates renal cortical COX-2 expression and
that COX-2 is a mediator of increased renin production in response to
inhibition of angiotension II production.
Zhang MZ, Wang JL, Cheng HF, Harris RC, McKanna JA. Cyclooxygenase-2 in rat nephron development. Am J Physiol (1997) 273:F994-1002
View abstract View in PubMed
The inducible second isoform of cyclooxygenase (COX-2) that mediates
inflammation also is expressed at low levels in normal adult rat kidneys
and is upregulated in response to noninflammatory stimuli (R. C. Harris,
J. A. McKanna, Y. Akai, H. R. Jacobson, R. N. DuBois, and M. D. Breyer, J.
Clin. Invest. 94: 2504-2510, 1994). Roles in morphogenesis are indicated
by reported teratogenicity of COX inhibitors and renal dysgenesis in COX-2
knockout mice (J. E. Dinchuk, B. D. Car, R. J. Focht, J. J. Johnston, B.
D. Jaffee, M. B. Covington, N. R. Contel, V. M. Eng, R. J. Collins, P. M.
Czerniak, A. G. Stewart, and J. M. Trzaskos, Nature 378: 406-409, 1995; S.
G. Morham, R. Lagenbach, C. D. Loftin, H. F. Tiano, N. Vouloumanos, J. C.
Jennette, J. F. Mahler, K. D. Kluckman, A. Ledford, C. A. Lee, and O.
Smithies. Cell 83: 473-482, 1995). Blots from developing rat kidneys
demonstrated that COX-2 mRNA and immunoreactive protein were present in
neonates, peaked in the 2nd and 3rd postnatal weeks and declined to adult
levels by the 3rd month. Immunolocalization and in situ hybridization
detected intense COX-2 immunoreactivity and mRNA in a subset of thick
ascending limb epithelial cells near the macula densa in each developing
nephron; after 2 wk the COX-2 gradually waned. These data demonstrate that
COX-2 expression is subject to normal developmental regulation and can be
sustained over extended periods; they also support the conclusion that
metabolites of COX-2 play important roles in the differentiation and early
functions of mammalian nephrons.
Harris RC, McKanna JA, Akai Y, Jacobson HR, Dubois RN, Breyer MD. Cyclooxygenase-2 is associated with the macula densa of rat kidney and
increases with salt restriction. J Clin Invest (1994) 94:2504-10
View abstract View in PubMed
The kidney is a rich source of prostaglandins. These eicosanoids, formed
by cyclooxygenase-dependent metabolism of arachidonic acid, are important
physiologic mediators of renal glomerular hemodynamics and tubular sodium
and water reabsorption. Two separate isoforms of cyclooxygenase (COX) have
now been identified: constitutive COX-1, encoded by a 2.8-kb mRNA, and
mitogen-activated COX-2, encoded by a 4.0-4.5-kb mRNA. COX-2 expression
increases during development and inflammation, but, except for brain,
constitutive expression is low. It has been generally accepted that
physiologic renal production of prostaglandins is mediated by COX-1.
However, in the absence of inflammation, low levels of COX-2 mRNA are also
detectable in the kidney. To examine the role of COX-2 in the kidney and
determine its intrarenal localization, we used a 1.3-kb cDNA probe
specific for the 3' untranslated region of rat COX-2 and COX-2-specific
antiserum. The COX-2-specific cDNA probe hybridized with a 4.4-kb
transcript in total RNA from adult rat kidney. Immunoblots of microsomes
isolated from kidney cortex and papilla indicated immunoreactive COX-2 in
both locations. In situ hybridization and immunohistochemistry indicated
that renal cortical COX-2 expression was localized to the macula densa of
the juxtaglomerular apparatus and to adjacent epithelial cells of the
cortical thick ascending limb of Henle. In addition, COX-2
immunoreactivity was detected in interstitial cells in the papilla. No
COX-2 message or immunoreactive protein was detected in arterioles,
glomeruli, or cortical or medullary collecting ducts. When animals were
chronically sodium restricted, the level of COX-2 in the region of the
macula densa increased threefold (from 0.86 +/0.08 to 2.52 +/0.43/mm2)
and the total area of the COX-2 immunoreactive cells in cortex increased
from 34 microns2/mm2 of cortex to 226 microns2/mm2 of cortex. The
intrarenal distribution of COX-2 and its increased expression in response
to sodium restriction suggest that in addition to its proposed role in
inflammatory and growth responses, this enzyme may play an important role
in the regulation of salt, volume, and blood pressure homeostasis.
|Service||Cost (Vanderbilt)||Cost (non-Vanderbilt)|
Click on the service name to request the service.
Additional content related to Immunohistochemistry
Last updated on 2009-07-08 Moderated by: