A simple treatment to significantly increase signal specificity in immunohistochemistry

Gong, Hua-Qin; Peng, Yi-Ben; Zou, Cheng; Wang, Donghui; Xu, Zhi-Hong; Bai, Shu-Nong

doi:10.1007/bf02914049

Cited by 15 publications

(10 citation statements)

References 13 publications

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“…Primary antibody directed against the first 42 N-terminal amino acids of AtPIP1;3 (Kammerloher et al ., 1994; Henzler et al ., 1999) was applied overnight at 4 °C. Slides were washed as described previously (Gong et al ., 2006). DyLight 549-conjugated rabbit anti-chicken secondary antibody was pre-absorbed with plant tissue extract (1:500 in BS) before it was applied for 2h at 37 °C.…”

Section: Methodsmentioning

confidence: 99%

Transpirational demand affects aquaporin expression in poplar roots

Laur

Hacke

2013

Journal of Experimental Botany

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Isohydric plants tend to maintain a water potential homeostasis primarily by controlling water loss via stomatal conductance. However, there is accumulating evidence that plants can also modulate water uptake in a dynamic manner. The dynamics of water uptake are influenced by aquaporin-mediated changes in root hydraulics. Most studies in this area have been conducted on herbaceous plants, and less is known about responses of woody plants. Here a study was conducted to determine how roots of hybrid poplar plants (Populus trichocarpa×deltoides) respond to a step change in transpirational demand. The main objective was to measure the expression of selected aquaporin genes and to assess how transcriptional responses correspond to changes in root water flow (Q R) and other parameters of water relations. A subset of plants was grown in shade and was subsequently exposed to a 5-fold increase in light level. Another group of plants was grown at ~95% relative humidity (RH) and was then subjected to lower RH while the light level remained unchanged. Both plant groups experienced a transient drop in stem water potentials. At 28h after the increase in transpirational demand, water potentials recovered. This recovery was associated with changes in the expression of PIP1 and PIP2 subfamily genes and an increase in Q R. Stomata of plants growing at high RH were larger and showed incomplete closure after application of abscisic acid. Since stomatal conductance remained high and unchanged in these plants, it is suggested that the recovery in water potential in these plants was largely driven by the increase in Q R.

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Section: Methodsmentioning

confidence: 99%

Transpirational demand affects aquaporin expression in poplar roots

Laur

Hacke

2013

Journal of Experimental Botany

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“…Cross sections (8 mm) were prepared using a microtome, mounted on slides, and dried overnight at 37°C. The next day, sections were dewaxed twice for 10 min each in Histochoice (SigmaAldrich), rehydrated through an ethanol series, rinsed three times for 10 min in PBS, and incubated for 45 min in a blocking solution (1.5% [w/v] Gly, 5% [w/v] bovine serum albumin, 0.1% [v/v] Tween 20 in PBS) as previously described (Gong et al, 2006). Primary antibodies against the first 42 N-terminal amino acids of AtPIP1;3 (Kammerloher et al, 1994;59-M EGKEEDVRVGANKFPERQPIGTSAQSDKDYKEPPPAPFFEP-39) and the highly conserved 10 amino acids of the C-terminal of PIP2 aquaporins (similar to Daniels et al, 1994;59-KALGSFRSNP-39) were included in this study.…”

Section: Immunolocalizationmentioning

confidence: 99%

“…Primary antibodies against the first 42 N-terminal amino acids of AtPIP1;3 (Kammerloher et al, 1994;59-M EGKEEDVRVGANKFPERQPIGTSAQSDKDYKEPPPAPFFEP-39) and the highly conserved 10 amino acids of the C-terminal of PIP2 aquaporins (similar to Daniels et al, 1994;59-KALGSFRSNP-39) were included in this study. Two secondary antibodies preabsorbed with plant tissue extract, DyLight 549 conjugated rabbit anti-chicken antibody (Fisher Scientific) and HiLyte Fluor 555 (AnaSpec) conjugated rabbit anti-mice antibody, were applied for 2 h at 37°C as previously described (Gong et al, 2006). Slides were rinsed several times and coverslipped with a synthetic resin (Permount; Fisher Scientific).…”

Section: Immunolocalizationmentioning

confidence: 99%

Uptake of Water via Branches Helps Timberline Conifers Refill Embolized Xylem in Late Winter

et al. 2014

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Xylem embolism is a limiting factor for woody species worldwide. Conifers at the alpine timberline are exposed to drought and freeze-thaw stress during winter, which induce potentially lethal embolism. Previous studies indicated that timberline trees survive by xylem refilling. In this study on Picea abies, refilling was monitored during winter and spring seasons and analyzed in the laboratory and in situ experiments, based on hydraulic, anatomical, and histochemical methods. Refilling started in late winter, when the soil was frozen and soil water not available for the trees. Xylem embolism caused up to 86.2% ± 3.1% loss of conductivity and was correlated with the ratio of closed pits. Refilling of xylem as well as recovery in shoot conductance started in February and corresponded with starch accumulation in secondary phloem and in the mesophyll of needles, where we also observed increasing aquaporin densities in the phloem and endodermis. This indicates that active, cellular processes play a role for refilling even under winter conditions. As demonstrated by our experiments, water for refilling was thereby taken up via the branches, likely by foliar water uptake. Our results suggest that refilling is based on water shifts to embolized tracheids via intact xylem, phloem, and parenchyma, whereby aquaporins reduce resistances along the symplastic pathway and aspirated pits facilitate isolation of refilling tracheids. Refilling must be taken into account as a key process in plant hydraulics and in estimating future effects of climate change on forests and alpine tree ecosystems.

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“…Next, samples were embedded, sectioned, dewaxed and rehydrated as described previously (Almeida-Rodriguez et al, 2011). Before the first immunoreaction, cross-sections were incubated for 45 min with blocking solution (BS; 1.5% (w/v) glycine, 5% (w/v) bovine serum albumin, 0.1% (v/v) Tween-20 in PBS) following the protocol of Gong et al (2006). Primary antibodies against the 42 N-terminal amino acids of AtPIP1;3 (Kammerloher et al, 1994) and the conserved 10 amino acids of the C-terminus of PIP2 AQPs (similar to Daniels et al, 1994) were included (see alignment in Fig.…”

Section: Immunolocalizationmentioning

confidence: 99%

Exploring Picea glauca aquaporins in the context of needle water uptake and xylem refilling

2014

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SummaryConifer needles have been reported to absorb water under certain conditions. Radial water movement across needle tissues is likely influenced by aquaporin (AQP) water channels.Foliar water uptake and AQP localization in Picea glauca needles were studied using physiological and microscopic methods. AQP expression was measured using quantitative real-time PCR. Members of the AQP gene family in spruce were identified using homology search tools.Needles of drought-stressed plants absorbed water when exposed to high relative humidity (RH). AQPs were present in the endodermis-like bundle sheath, in phloem cells and in the transfusion parenchyma of needles. Up-regulation of AQPs in high RH coincided with embolism repair in stem xylem. The present study also provides the most comprehensive functional and phylogenetic analysis of spruce AQPs to date. Thirty putative complete AQP sequences were found.Our findings are consistent with the hypothesis that AQPs facilitate radial water movement from the needle epidermis towards the vascular tissue. Foliar water uptake may occur in late winter when needles are covered by melting snow and may provide a water source for embolism repair before the beginning of the growing season.

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A simple treatment to significantly increase signal specificity in immunohistochemistry

Cited by 15 publications

References 13 publications

Transpirational demand affects aquaporin expression in poplar roots

Transpirational demand affects aquaporin expression in poplar roots

Uptake of Water via Branches Helps Timberline Conifers Refill Embolized Xylem in Late Winter

Exploring Picea glauca aquaporins in the context of needle water uptake and xylem refilling

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