Role of microwave heating in antigen retrieval in cryosections of the formalin-fixed mammalian tissues

Brzica, Hrvoje; Breljak, Davorka; Vrhovac, Ivana; Sabolić, Ivan

doi:10.5772/20319

Cited by 6 publications

(13 citation statements)

References 43 publications

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“…Further steps, which included tissue cryosectioning, an antigen retrieval protocol, and incubation with primary and secondary antibodies, were described in detail in our previous publications [ 2 , 45 , 46 ]. The optimal protocol to retrieve mSglt1 and Na + /K + -ATPase epitopes included a microwave oven heating in 10 mM citrate buffer, pH 6 [ 12 ]. Dilutions of primary antibodies were 1:25–1:300 for our non-commercial mSglt1-Ab, 1:100 for commercial anti-SGLT1 antibodies, and 1:100 for Na/K-ATPase-Ab.…”

Section: Methodsmentioning

confidence: 99%

Expression profiling and immunolocalization of Na+-d-glucose-cotransporter 1 in mice employing knockout mice as specificity control indicate novel locations and differences between mice and rats

Madunić

Breljak

Karaica

et al. 2017

Pflugers Arch - Eur J Physiol

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The expression and localization of sodium-d-glucose cotransporter SGLT1 (SLC5A1), which is involved in small intestinal glucose absorption and renal glucose reabsorption, is of high biomedical relevance because SGLT1 inhibitors are currently tested for antidiabetic therapy. In human and rat organs, detailed expression profiling of SGLT1/Sglt1 mRNA and immunolocalization of the transporter protein has been performed. Using polyspecific antibodies and preabsorption with antigenic peptide as specificity control, in several organs, different immunolocalizations of SGLT1/Sglt1 between human and rat were obtained. Because the preabsorption control does not exclude cross-reactivity with similar epitopes, some localizations remained ambiguous. In the present study, we performed an immunocytochemical localization of Sglt1 in various organs of mice. Specificities of the immunoreactions were evaluated using antibody preabsorption with the Sglt1 peptide and the respective organs of Sglt1 knockout mice. Because staining in some locations was abolished after antibody preabsorption but remained in the knockout mice, missing staining in knockout mice was used as specificity criterion. The immunolocalization in mouse was identical or similar to rat in many organs, including small intestine, liver, and kidney. However, the male-dominant renal Sglt1 protein expression in mice differed from the female-dominant expression in rats, and localization in lung, heart, and brain observed in rats was not detected in mice. In mice, several novel locations of Sglt1, e.g., in eyes, tongue epithelial cells, pancreatic ducts, prostate, and periurethral glands were detected. Using end-point and quantitative RT-PCR in various organs, different Sglt1 expression in mice and rats was confirmed.Electronic supplementary materialThe online version of this article (doi:10.1007/s00424-017-2056-1) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

Expression profiling and immunolocalization of Na+-d-glucose-cotransporter 1 in mice employing knockout mice as specificity control indicate novel locations and differences between mice and rats

Madunić

Breljak

Karaica

et al. 2017

Pflugers Arch - Eur J Physiol

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“…The optimal antigen retrieval conditions included heating of 4-m cryosections in a microwave oven at 800 W (four cycles, 5 min each) in 10 mM citrate buffer at pH 3 (NaDC3, AQP1, SGLT1, and V-ATPase), pH 6 (OAT2 and OAT3), or pH 8 (OAT1), whereas the optimal retrieval conditions for revealing Na ϩ -K ϩ -ATPase epitopes included pretreatment of cryosections with xylol and graded concentrations of ethanol followed by microwave heating in 10 mM citrate buffer at pH 6. The respective protocols have been previously described in detail (8).…”

Section: Methodsmentioning

confidence: 99%

“…For single staining, tissue cryosections were incubated overnight in a refrigerator in PBS containing an optimized concentration of the respective Ab (1:25-1:200) followed by a rinse in Triton X-100containing PBS buffers, incubation for 1 h at room temperature with GARCY3, and a rinse in Triton X-100-containing PBS buffers (3,8,45). To test the Ab specificity for the immunizing peptide, the respective primary Abs were blocked with the corresponding immunizing peptides (final concentrations: 0.5 mg/ml) for 4 h at room temperature before use in immunofluorescence assays.…”

Section: Methodsmentioning

confidence: 99%

Distribution of organic anion transporters NaDC3 and OAT1-3 along the human nephron

Breljak

Ljubojević

Hagos

et al. 2016

American Journal of Physiology-Renal Physiology

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The initial step in renal secretion of organic anions (OAs) is mediated by transporters in the basolateral membrane (BLM). Contributors to this process are primary active Na(+)-K(+)-ATPase (EC 3.6.3.9), secondary active Na(+)-dicarboxylate cotransporter 3 (NaDC3/SLC13A3), and tertiary active OA transporters (OATs) OAT1/SLC22A6, OAT2/SLC22A7, and OAT3/SLC22A8. In human kidneys, we analyzed the localization of these transporters by immunochemical methods in tissue cryosections and isolated membranes. The specificity of antibodies was validated with human embryonic kidney-293 cells stably transfected with functional OATs. Na(+)-K(+)-ATPase was immunolocalized to the BLM along the entire human nephron. NaDC3-related immunostaining was detected in the BLM of proximal tubules and in the BLM and/or luminal membrane of principal cells in connecting segments and collecting ducts. The thin and thick ascending limbs, macula densa, and distal tubules exhibited no reactivity with the anti-NaDC3 antibody. OAT1-OAT3-related immunostaining in human kidneys was detected only in the BLM of cortical proximal tubules; all three OATs were stained more intensely in S1/S2 segments compared with S3 segment in medullary rays, whereas the S3 segment in the outer stripe remained unstained. Expression of NaDC3, OAT1, OAT2, and OAT3 proteins exhibited considerable interindividual variability in both male and female kidneys, and sex differences in their expression could not be detected. Our experiments provide a side-by-side comparison of basolateral transporters cooperating in renal OA secretion in the human kidney.

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“…Leica CM 1850 cryostat (Leica instruments GmbH, Nussloch, Germany) was used to cut 4 μm thick cryosections, which were collected on SuperFrost Plus microscope slides (Thermo Scientific, Gerhard Menzel GmbH, Braunschweig, Germany), dried at room temperature for 2 h, and stored refrigerated until use. In preliminary studies, cryosections were tested for optimal antigen retrieval protocol; 15 microwave heating in citrate buffer pH 6 yielded the best staining intensity and was used for presenting both OCTs. In short, cryosections underwent the following steps: rehydration in PBS for 15 min, heating in citrate buffer pH 6 in a microwave oven at 800 W for 20 min, washing in PBS (3 × 5 min), incubation in 0.5% Triton-X-100 (in PBS) for 15 min and 2% Triton-X-100 (in PBS) for 30 min, washing in PBS (3 × 5 min), incubation in bovine serum albumin (1% solution in PBS) for 30 min, incubation in primary antibody at 4 °C overnight, incubation in secondary antibody at room temperature for 60 min, and washing in 0.1% Triton-X-100 (10 min) and PBS (3 × 5 min).…”

Section: ■ Introductionmentioning

confidence: 99%

Kidney Transplantation Down-Regulates Expression of Organic Cation Transporters, Which Translocate β-Blockers and Fluoroquinolones

et al. 2013

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Kidney transplanted patients are often treated with immunosuppressive, antihypertensive, and antibiotic drugs such as cyclosporine A (CsA), β-blockers, and fluoroquinolones, respectively. Organic cation transporters (OCT) expressed in the basolateral membrane of proximal tubules represent an important drug excretion route. In this work, the renal expression of OCT after syngeneic and allogeneic kidney transplantation in rats with or without CsA immunosuppression was studied. Moreover, the interactions of CsA, β-blockers (pindolol/atenolol), and fluoroquinolones (ofloxacin/norfloxacin) with rOCT1, rOCT2, hOCT1, and hOCT2 in stably transfected HEK293-cells were studied. Kidney transplantation was associated with reduced expression of rOCT1, while rOCT2 showed only reduced expression after allogeneic transplantation. All drugs interacted subtype- and species-dependently with OCT. However, only atenolol, pindolol, and ofloxacin were transported by hOCT2, the main OCT in human kidneys. While CsA is not an OCT substrate, it exerts a short-term effect on OCT activity, changing their affinity for some substrates. In conclusion, appropriate drug dosing in transplanted patients is difficult partly because OCT are down-regulated and because concomitant CsA treatment may influence the affinity of the transporters. Moreover, drug-drug competition at the transporter can also alter drug excretion rate.

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Role of microwave heating in antigen retrieval in cryosections of the formalin-fixed mammalian tissues

Cited by 6 publications

References 43 publications

Expression profiling and immunolocalization of Na+-d-glucose-cotransporter 1 in mice employing knockout mice as specificity control indicate novel locations and differences between mice and rats

Expression profiling and immunolocalization of Na+-d-glucose-cotransporter 1 in mice employing knockout mice as specificity control indicate novel locations and differences between mice and rats

Distribution of organic anion transporters NaDC3 and OAT1-3 along the human nephron

Kidney Transplantation Down-Regulates Expression of Organic Cation Transporters, Which Translocate β-Blockers and Fluoroquinolones

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