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Section Pretreatment, Epitope Demasking, and Methods for Dealing with Unwanted Staining
Published in Lars-Inge Larsson, Immunocytochemistry: Theory and Practice, 2020
Most immunocytochemists employ either phosphate-buffered saline (PBS; 0.01 M sodium phosphate buffer containing 0.15 M NaCl with the pH adjusted to 7.4; molarities of the phosphate buffer may vary in different applications) or tris-buffered saline (TBS; 0.01 to 0.05 M tris buffer containing 0.15 M NaCl with the pH adjusted to 7.4 to 7.6). As the standard DAB reaction is carried out in tris buffer, many immunoperoxidase immunocytochemists employ TBS, while immunofluorescence microscopists frequently use PBS. Clear differences between the two buffers on immune staining have never been documented. However, Grossi and von Mayersbach noted that tris buffers extracted nucleic acids from unfixed or methanol-fixed cryostat sections.16 In immunogold staining applications, many workers prefer to use higher pH values (believed to diminish aggregation of gold particles); in the GLAD method, 0.05 M tris buffer, pH 7.9 to 8.2, containing 0.15 M NaCl (and added gold particle stabilizers such as polyethylene glycol or BSA) is recommended.37 Similar buffers may be employed in all gold staining techniques (cf. Appendix).
Lectin
Published in Masahiko Mori, Histochemistry of the Salivary Glands, 2019
Leathem and Atkins12 stated that the direct antibody method resulted in excellent findings with high specificity and high intensity staining. They also stated that the direct antibody method was eight times more sensitive than the direct lectin-conjugate technique, and the indirect antibody method was 10 to 20 times more sensitive than the direct lectin-conjugate method. Lectins require the presence of metal ions for binding, and phosphate buffer salts may precipitate these ions; therefore, Tris-buffered saline (TBS) solution should be used.
Improved therapeutic index of an acidic pH-selective antibody
Published in mAbs, 2022
Peter S. Lee, Katherine G. MacDonald, Evan Massi, Pamela V. Chew, Christine Bee, Padma Perkins, Bryant Chau, Kent Thudium, Jack Lohre, Pradyot Nandi, Ekaterina G. Deyanova, Ishita Barman, Olafur Gudmundsson, Gavin Dollinger, Tim Sproul, John J. Engelhardt, Pavel Strop, Arvind Rajpal
SPR was used to determine binding parameters for various Ipi variants to human and cyno CTLA-4 with a BIACORE® T200 SPR spectrometer (Biacore AB, Uppsala, Sweden) at 37°C. Fabs of Ipi variants were captured on a BIACORE® CM4 chip with immobilized anti-human kappa polyclonal capture antibody. Monomeric human or cyno CTLA-4 was flowed as analyte with up to 2 μM top concentration. Between cycles, the capture surface was regenerated with 75 mM phosphoric acid. Running buffers were HEPES buffered saline for pH 7.4 experiments and Bis-Tris buffered saline for experiments with lower pH. All buffers were supplemented with 0.05% Tween-20 and 1 g/L BSA. Double-referenced sensorgrams were fitted to a 1:1 Langmuir binding model with mass transport to determine equilibrium dissociation constants (KD), as well as association (ka) and dissociation (kd) rate constants where appropriate.
BRAF testing in a South African cohort of MLH1 deficient endometrial carcinomas: lessons learnt
Published in Southern African Journal of Gynaecological Oncology, 2021
Immunohistochemistry was performed on 4 μm deparaffinised sections using MLH1 (Novocastra, UK, Clone ES05, 1:50), PMS2 (Novocastra, UK, Clone MOR4G, 1:50), MSH2 (Novocastra, UK, Clone 25D12, 1:50), MSH6 (Novocastra, UK, PU29, 1:50) and BRAF V600E (Clone VE1). Tissue sections were cut with a microtome from paraffin-embedded blocks. The cut sections were floated onto slides that were then dried in an incubator at 60°C overnight. The immunohistochemical stains have all been optimised according to departmental standard operating procedure and the manufacturer instructions for use on an automated staining machine (DAKO Autostainer Link 48, Denmark). Staining of tissue sections includes the treatment of primary antibody serum. Usage of a mouse linker resulted in increased expression of antigens. Tissue sections were washed with Tris buffered saline (TBS) at pH 7.6. The EnVision™ FLEX target Retrieval Solution, High pH, was used for antigen retrieval. The chromogen 3,3’ Diaminobenzidine hydrochloride solution (DAB, Sigma) was used, which produced a brown pigment. Meyer’s haematoxylin was the counterstain utilised. Tissue sections, in the presence of positive internal controls such as endothelial cells, stromal cells and lymphocytes, were assessed for retention of staining in tumour nuclei or an absence/loss of staining in tumour cells.
Kv7 channel inhibition increases hypoxic pulmonary vasoconstriction in endotoxemic mouse lungs
Published in Experimental Lung Research, 2020
Maurizio Turzo, Fabian A. Spöhr, Lasitschka Felix, Markus A. Weigand, Cornelius J. Busch
Lungs of mice with and without endotoxemia were fixed in paraformaldehyde (n = 4 each group). Immunohistology was performed on 2-μm paraffin-embedded sections, using standard avidin-biotin, anti-alkaline phosphatase technique (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s instructions. Tris-buffered saline supplemented with 0.2% bovine serum albumin (Biotrend, Cologne, Germany), was used as buffer. Primary antibody dilutions of polyclonal rabbit anti-KCNE3, 1/50 (Abcam, ab86203, Cambridge, UK) and an isotype- and concentration-matched rabbit control Ig (Dianova, Hamburg, Germany) were prepared in this buffer and incubated for 1 h at room temperature. A biotinylated donkey anti-rabbit IgG Ab, 1/100 (Jackson ImmunoResearch, Newmarket, UK), was used as a secondary reagent (30 min at room temperature). Naphthol AS-biphosphate (Sigma-Aldrich) with New-fuchsin (Merck) was used as the substrate for alkaline phosphatase. Slides were counterstained with hematoxylin (Sigma-Aldrich). Magenta positive staining (corresponding to KCNE3 positive staining) was defined as the area of interest and was quantified (BX63 Upright Microscope; cellSens Dimension 1.17 software, Olympus, Tokyo, Japan).